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1
Balasuriya, Nileeka, McShane McKenna, Xuguang Liu, Shawn Li, and Patrick O’Donoghue. "Phosphorylation-Dependent Inhibition of Akt1." Genes 9, no. 9 (September 7, 2018): 450. http://dx.doi.org/10.3390/genes9090450.
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Protein kinase B (Akt1) is a proto-oncogene that is overactive in most cancers. Akt1 activation requires phosphorylation at Thr308; phosphorylation at Ser473 further enhances catalytic activity. Akt1 activity is also regulated via interactions between the kinase domain and the N-terminal auto-inhibitory pleckstrin homology (PH) domain. As it was previously difficult to produce Akt1 in site-specific phosphorylated forms, the contribution of each activating phosphorylation site to auto-inhibition was unknown. Using a combination of genetic code expansion and in vivo enzymatic phosphorylation, we produced Akt1 variants containing programmed phosphorylation to probe the interplay between Akt1 phosphorylation status and the auto-inhibitory function of the PH domain. Deletion of the PH domain increased the enzyme activity for all three phosphorylated Akt1 variants. For the doubly phosphorylated enzyme, deletion of the PH domain relieved auto-inhibition by 295-fold. We next found that phosphorylation at Ser473 provided resistance to chemical inhibition by Akti-1/2 inhibitor VIII. The Akti-1/2 inhibitor was most effective against pAkt1T308 and showed four-fold decreased potency with Akt1 variants phosphorylated at Ser473. The data highlight the need to design more potent Akt1 inhibitors that are effective against the doubly phosphorylated and most pathogenic form of Akt1.
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BARNETT, Stanley F., Deborah DEFEO-JONES, Sheng FU, Paula J. HANCOCK, Kathleen M. HASKELL, Raymond E. JONES, Jason A. KAHANA, et al. "Identification and characterization of pleckstrin-homology-domain-dependent and isoenzyme-specific Akt inhibitors." Biochemical Journal 385, no. 2 (January 7, 2005): 399–408. http://dx.doi.org/10.1042/bj20041140.
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We developed a high-throughput HTRF (homogeneous time-resolved fluorescence) assay for Akt kinase activity and screened approx. 270000 compounds for their ability to inhibit the three isoforms of Akt. Two Akt inhibitors were identified that exhibited isoenzyme specificity. The first compound (Akt-I-1) inhibited only Akt1 (IC50 4.6 μM) while the second compound (Akt-I-1,2) inhibited both Akt1 and Akt2 with IC50 values of 2.7 and 21 μM respectively. Neither compound inhibited Akt3 nor mutants lacking the PH (pleckstrin homology) domain at concentrations up to 250 μM. These compounds were reversible inhibitors, and exhibited a linear mixed-type inhibition against ATP and peptide substrate. In addition to inhibiting kinase activity of individual Akt isoforms, both inhibitors blocked the phosphorylation and activation of the corresponding Akt isoforms by PDK1 (phosphoinositide-dependent kinase 1). A model is proposed in which these inhibitors bind to a site formed only in the presence of the PH domain. Binding of the inhibitor is postulated to promote the formation of an inactive conformation. In support of this model, antibodies to the Akt PH domain or hinge region blocked the inhibition of Akt by Akt-I-1 and Akt-I-1,2. These inhibitors were found to be cell-active and to block phosphorylation of Akt at Thr308 and Ser473, reduce the levels of active Akt in cells, block the phosphorylation of known Akt substrates and promote TRAIL (tumour-necrosis-factor-related apoptosis-inducing ligand)-induced apoptosis in LNCap prostate cancer cells.
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Carón, Rubén W., Adly Yacoub, Min Li, Xiaoyu Zhu, Clint Mitchell, Young Hong, William Hawkins, et al. "Activated forms of H-RAS and K-RAS differentially regulate membrane association of PI3K, PDK-1, and AKT and the effect of therapeutic kinase inhibitors on cell survival." Molecular Cancer Therapeutics 4, no. 2 (February 1, 2005): 257–70. http://dx.doi.org/10.1158/1535-7163.257.4.2.
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Abstract The abilities of mutated active RAS proteins to modulate cell survival following exposure to ionizing radiation and small molecule kinase inhibitors were examined. Homologous recombination in HCT116 cells to delete the single allele of K-RAS D13 resulted in a cell line that exhibited an ∼75% reduction in basal extracellular signal-regulated kinase 1/2, AKT, and c-jun-NH2-kinase 1/2 activity. Transfection of cells lacking K-RAS D13 with H-RAS V12 restored extracellular signal-regulated kinase 1/2 and AKT activity to basal levels but did not restore c-jun-NH2-kinase 1/2 phosphorylation. In cells expressing H-RAS V12, radiation caused prolonged intense activation of AKT. Inhibition of H-RAS V12 function, blockade of phosphatidylinositol 3-kinase (PI3K) function using small interfering RNA/small-molecule inhibitors, or expression of dominant-negative AKT abolished radiation-induced AKT activation, and radiosensitized these cells. Inhibition of PI3K function did not significantly radiosensitize parental HCT116 cells. Inhibitors of the AKT PH domain including perifosine, SH-(5, 23-25) and ml-(14-16) reduced the plating efficiency of H-RAS V12 cells in a dose-dependent fashion. Inhibition of AKT function using perifosine enhanced radiosensitivity in H-RAS V12 cells, whereas the SH and ml series of AKT PH domain inhibitors failed to promote radiation toxicity. In HCT116 H-RAS V12 cells, PI3K, PDK-1, and AKT were membrane associated, whereas in parental cells expressing K-RAS D13, only PDK-1 was membrane bound. In H-RAS V12 cells, membrane associated PDK-1 was phosphorylated at Y373/376, which was abolished by the Src family kinase inhibitor PP2. Inhibition of PDK-1 function using the PH domain inhibitor OSU-03012 or using PP2 reduced the plating efficiency of H-RAS V12 cells and profoundly increased radiosensitivity. OSU-03012 and PP2 did not radiosensitize and had modest inhibitory effects on plating efficiency in parental cells. A small interfering RNA generated against PDK1 also radiosensitized HCT116 cells expressing H-RAS V12. Collectively, our data argue that molecular inhibition of AKT and PDK-1 signaling enhances the radiosensitivity of HCT116 cells expressing H-RAS V12 but not K-RAS D13. Small-molecule inhibitory agents that blocked stimulated and/or basal PDK-1 and AKT function profoundly reduced HCT116 cell survival but had variable effects at enhancing tumor cell radiosensitivity.
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NA, Byoung-Kuk, Bhaskar R. SHENAI, Puran S. SIJWALI, Youngchool CHOE, Kailash C. PANDEY, Ajay SINGH, Charles S. CRAIK, and Philip J. ROSENTHAL. "Identification and biochemical characterization of vivapains, cysteine proteases of the malaria parasite Plasmodium vivax." Biochemical Journal 378, no. 2 (March 1, 2004): 529–38. http://dx.doi.org/10.1042/bj20031487.
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Cysteine proteases play important roles in the life cycles of malaria parasites. Cysteine protease inhibitors block haemoglobin hydrolysis and development in Plasmodium falciparum, suggesting that the cysteine proteases of this major human pathogen, termed falcipains, are appropriate therapeutic targets. To expand our understanding of plasmodial proteases to Plasmodium vivax, the other prevalent human malaria parasite, we identified and cloned genes encoding the P. vivax cysteine proteases, vivapain-2 and vivapain-3, and functionally expressed the proteases in Escherichia coli. The vivapain-2 and vivapain-3 genes predicted papain-family cysteine proteases, which shared a number of unusual features with falcipain-2 and falcipain-3, including large prodomains and short N-terminal extensions on the catalytic domain. Recombinant vivapain-2 and vivapain-3 shared properties with the falcipains, including acidic pH optima, requirements for reducing conditions for activity and hydrolysis of substrates with positively charged residues at P1 and Leu at P2. Both enzymes hydrolysed native haemoglobin at acidic pH and the erythrocyte cytoskeletal protein 4.1 at neutral pH, suggesting similar biological roles to the falcipains. Considering inhibitor profiles, the vivapains were inhibited by fluoromethylketone and vinyl sulphone inhibitors that also inhibited falcipains and have demonstrated potent antimalarial activity.
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Miyamoto, Seiji, Atsushi Iwasa, Masao Ishii, Kenji Okajima, and Yu-ichi Kamikubo. "Purification and Characterization of Factor VII Inhibitor Found in a Patient with Life Threatening Bleeding." Thrombosis and Haemostasis 83, no. 01 (2000): 60–64. http://dx.doi.org/10.1055/s-0037-1613758.
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SummaryWe recently observed a patient with acquired inhibitor-induced F.VII deficiency whose plasma level of F.VII was < 1.0%. However, the biochemical nature of the inhibitor has not yet been clarified. In the present study, we purified the F.VII inhibitor from the patient’s plasma by using activated F.VII (F.VIIa)-conjugated gel and characterized the inhibitor. The results showed that the inhibitor comprised two kinds of antibodies: one was eluted with EDTA (antibody 1) and the other with glycine-HCl buffer (pH 2.3) (antibody 2) from the F.VIIa affinity gel. SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting analysis of these inhibitors demonstrated that both antibodies had features of immunoglobulin G1 (IgG1) with κ and λ-light chains. Antibody 1 bound to the immobilized F.VIIa with a high affinity in the presence of calcium ion, while antibody 2 bound to the F.VIIa very weakly and the binding was independent of calcium ion. Immunoblotting analysis demonstrated that antibody 1 bound to the light chain of F.VIIa after reduction with 2-mercaptoethanol, while it did not react with either the γ carboxyglutamic acid (Gla)-domainless light chain of F.VIIa or the heavy chain with the protease domain. Antibody 1 markedly inhibited the activity of tissue factor-F.VIIa complex. Based on these observations, it is suggested that F.VIIa autoantibody (antibody 1) recognizes the calcium-dependent conformation within or near the Gla domain and inhibits F.VIIa activity by interacting with the light chain.
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Königs, Christoph, Christoph Kessel, Sabine Scholz, Susanne Stumpf, Manuela Krause, Thomas Klingebiel, and Wolfhart Kreuz. "Epitope Mapping of Inhibitors in Acquired Hemophilia by Phage Display." Blood 106, no. 11 (November 16, 2005): 3202. http://dx.doi.org/10.1182/blood.v106.11.3202.3202.
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Abstract In acquired hemophilia polyclonal autoantibodies (inhibitors) to coagulation factor VIII are generated. Inhibitors functionally interfere with interaction of molecules involved in the coagulation cascade for example the interaction of FVIII and the von Willebrand factor. Inhibitors in acquired hemophilia have been reported to bind either to the A2 or to C2 domain of FVIII. Random peptide phage display libraries were screened with patient plasma to identify small peptides mimicking inhibitor epitopes. Peptide sequences were used to map the inhibitor epitope to the surface of FVIII. The phage libraries included libraries with linear or cyclic 7mer or 12mer inserts. Biopanning was performed including positive selections and also negative selections to deplete irrelevant binders. Phages bound to inhibitors were eluted by pH shift or by competition with FVIII. Single phage clones were tested for their specificity for inhibitor positive plasma by ELISA. For specific binders, the sequence of the inserts was identified by DNA sequencing. For one patient 33 phage clones were analysed from three libraries and 28 peptide sequences mimicking the inhibitor epitopemimotopes - were determined. The peptide sequences map to two conformational epitopes on the A2 and A1 domain of FVIII. Clusters of six and four amino acids are the proposed binding regions on the A2 and A1 domain respectively. The matching conformational motifs contain PPLRQ and PPLS. Synthetic peptides corresponding to the sequence displayed on phage were generated and binding of inhibitors to peptides was confirmed by different ELISA formats. Currently, functional studies with synthetic peptides are being performed to further analyse the epitopes. Additionally, more patient samples are being screened to analyse further epitopes and understand immuno dominant regions of FVIII in acquired haemophilia and compare them to the immune response in hemophilia A. The mimotopes isolated could be a basis for the development of ligand effector conjugates to target inhibitor specific B cells.
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Kimura, Shinya, Haruna Naito, Hidekazu Segawa, Junya Kuroda, Takeshi Yuasa, Kiyoshi Sato, Asumi Yokota, et al. "NS-187, a potent and selective dual Bcr-Abl/Lyn tyrosine kinase inhibitor, is a novel agent for imatinib-resistant leukemia." Blood 106, no. 12 (December 1, 2005): 3948–54. http://dx.doi.org/10.1182/blood-2005-06-2209.
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Although the Abelson (Abl) tyrosine kinase inhibitor imatinib mesylate has improved the treatment of breakpoint cluster region–Abl (Bcr-Abl)–positive leukemia, resistance is often reported in patients with advanced-stage disease. Although several Src inhibitors are more effective than imatinib and simultaneously inhibit Lyn, whose overexpression is associated with imatinib resistance, these inhibitors are less specific than imatinib. We have identified a specific dual Abl-Lyn inhibitor, NS-187 (elsewhere described as CNS-9), which is 25 to 55 times more potent than imatinib in vitro. NS-187 is also at least 10 times as effective as imatinib in suppressing the growth of Bcr-Abl–bearing tumors and markedly extends the survival of mice bearing such tumors. The inhibitory effect of NS-187 extends to 12 of 13 Bcr-Abl proteins with mutations in their kinase domain but not to T315I. NS-187 also inhibits Lyn without affecting the phosphorylation of Src, Blk, or Yes. These results suggest that NS-187 may be a potentially valuable novel agent to combat imatinib-resistant Philadelphia-positive (Ph+) leukemia.
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Kasap, Corynn, Nicholas Hertz, Debora Makino, Kevan Shokat, John Kuriyan, and Neil P. Shah. "Beyond the Gatekeeper: Imatinib- and Dasatinib-Resistant BCR-ABL/F317 Mutations Confer Cross-Resistance to VX-680 but Are Sensitive to a Structural Derivative of VX-680." Blood 112, no. 11 (November 16, 2008): 725. http://dx.doi.org/10.1182/blood.v112.11.725.725.
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Abstract The management of chronic phase CML has been revolutionized by selective ABL tyrosine kinase inhibitor (TKI) therapy. Despite the effectiveness of these targeted agents, long-term control of blast phase CML and Ph+ ALL has been elusive, where the majority of patients relapse within 6–12 months. For blast phase CML and Ph+ ALL, two TKIs are currently approved: imatinib and dasatinib. While head-to-head comparisons of these agents have not been performed, it is generally believed that dasatinib is the more active agent for these phases of disease. In most cases, loss of response to these agents is driven by BCR-ABL kinase domain mutations. While more than 70 mutations have been associated with clinical resistance to imatinib, dasatinib appears vulnerable primarily to five mutations: V299L, T315A, T315I, F317I, and F317L. Of these, T315I and F317L are cross-resistant to imatinib. For the achievement of long-term remissions in blast phase CML and Ph+ ALL, a combination of TKIs that can collectively suppress all resistant BCR-ABL kinase domain mutations holds therapeutic promise. The BCR-ABL/T315I mutation, which confers a high degree of resistance to all approved BCR-ABL TKIs, has been referred to as a “molecular gatekeeper”, as it restricts access to a deeper hydrophobic pocket within the ABL kinase domain and makes an important stabilizing H-bond with imatinib, dasatinib and nilotinib. The Aurora kinase inhibitor VX-680 was the first compound to have activity against BCR-ABL/T315I in vitro, as well as clinically. To determine the promise of a kinase inhibitor combination of dasatinib and VX-680, we assessed the activity of VX-680 against the five dasatinib-resistant mutations using a cell-based flow cytometric assay of BCR-ABL kinase activity. While three mutants are sensitive, mutations at F317 demonstrated a high degree of resistance. We tested a number of other Aurora kinase inhibitors of different chemotypes and found that each of these had similar difficulty at inhibiting the kinase activity of BCR-ABL/F317 mutants. Based upon the co-crystal structure of VX-680 complexed with ABL, we have performed structure-activity relationship studies of 12 VX-680 scaffold derivatives, and have successfully identified structural modifications that increase kinase inhibitory activity against F317 mutants. Moreover, one of these derivatives increases the selectivity for ABL relative to Aurora kinases, which may help reduce the likelihood of suppressing normal hematopoiesis, a dose-limiting toxicity of Aurora kinase inhibitors that may substantially limit their effectiveness for the management of hematologic malignancies such as blast phase CML and Ph+ ALL. Lastly, we have performed structural studies of ABL/F317 mutants complexed with select VX-680 derivatives in an effort to understand how F317 mutations confer resistance to a broad range of ABL and Aurora kinase inhibitors. Interestingly, a recent study reported the successful selection of Aurora kinase inhibitor-resistant clones derived from a human colon cancer cell line (Girdler et al, 2008). While no resistance-conferring mutations were isolated at L154, the Aurora kinase gatekeeper residue, mutations were detected at Y156 in Aurora B, which corresponds to F317 in ABL. Aurora B Y156 mutations were found to confer resistance to a number of Aurora kinase inhibitors, including VX-680. As Aurora kinase inhibitors are being studied in a variety of non-hematologic malignancies, there is an increasing need to understand and overcome the mechanisms whereby mutations at this residue confer resistance to these agents. It is hoped that our studies will lead not only to the development of an effective adjunctive kinase inhibitor for the treatment of blast phase CML and Ph+ ALL, but will also shed light on the growing problem of resistance conferred by mutations at residues that correspond to BCR-ABL/F317 in other kinases.
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Liao, Maofu, and Margaret Kielian. "Domain III from class II fusion proteins functions as a dominant-negative inhibitor of virus membrane fusion." Journal of Cell Biology 171, no. 1 (October 10, 2005): 111–20. http://dx.doi.org/10.1083/jcb.200507075.
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Alphaviruses and flaviviruses infect cells through low pH-dependent membrane fusion reactions mediated by their structurally similar viral fusion proteins. During fusion, these class II viral fusion proteins trimerize and refold to form hairpin-like structures, with the domain III and stem regions folded back toward the target membrane-inserted fusion peptides. We demonstrate that exogenous domain III can function as a dominant-negative inhibitor of alphavirus and flavivirus membrane fusion and infection. Domain III binds stably to the fusion protein, thus preventing the foldback reaction and blocking the lipid mixing step of fusion. Our data reveal the existence of a relatively long-lived core trimer intermediate with which domain III interacts to initiate membrane fusion. These novel inhibitors of the class II fusion proteins show cross-inhibition within the virus genus and suggest that the domain III–core trimer interaction can serve as a new target for the development of antiviral reagents.
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NAGASE, Hideaki, Ko SUZUKI, Tim E. CAWSTON, and K. BREW. "Involvement of a region near valine-69 of tissue inhibitor of metalloproteinases (TIMP)-1 in the interaction with matrix metalloproteinase 3 (stromelysin 1)." Biochemical Journal 325, no. 1 (July 1, 1997): 163–67. http://dx.doi.org/10.1042/bj3250163.
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Tissue inhibitors of metalloproteinases (TIMPs) inhibit matrix metalloproteinases (MMPs) by forming a 1:1 stoichiometric complex, but the inhibition mechanism of these inhibitors is not known. Here we have investigated the reactive site of TIMP-1 by its proteinase susceptibility before and after forming a complex with MMP-3 (stromelysin 1). When TIMP-1 was allowed to react with human neutrophil elastase, its inhibitory activity was destroyed. This resulted from cleavage of the Val69–Cys70 bond. However, cleavage of this bond by neutrophil elastase was prevented when TIMP-1 formed a complex with the catalytic domain of MMP-3, and full TIMP-1 activity was restored after dissociation of the complex at pH 3.0 in the presence of EDTA. These results indicate that the region around Val69 closely associates with an active MMP. The three-dimensional structure of the N-terminal domain of TIMP-2 elucidated by NMR studies [Williamson, Martorell, Carr, Murphy, Docherty, Freedman and Feeney (1994) Biochemistry 33, 11745–11759] reveals that Val69 and Cys70 form part of an extended ridge that also includes the N-terminal section of the inhibitor. This region is probably involved in the interaction with the catalytic domains of MMPs.
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Okabe, Seiichi, Tetsuzo Tauchi, and Kazuma Ohyashiki. "GDC-0449, the Small Molecule Inhibitor of Hedgehog-Gli Pathway for the Treatment of BCR-ABL Positive Leukemia Cells and In Combination with Dasatinib." Blood 116, no. 21 (November 19, 2010): 2136. http://dx.doi.org/10.1182/blood.v116.21.2136.2136.
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Abstract Abstract 2136 Imatinib has shown clinical efficacy against Philadelphia chromosome (Ph) positive leukemia cells and it is now the standard care for initial therapy. However, recent studies reported imatinib are not effective in quiescent primitive chronic myeloid leukemia (CML) stem cells. Moreover, many Ph-positive leukemia patients develop resistance or fail to respond to imatinib by mutation in the ABL kinase domain in clinically. These results indicated that alternative combination therapy such as BCR-ABL targeting tyrosine kinase inhibitors (TKIs) and nontoxic agents are required to cure the Ph positive leukemia patients. Hedgehog (Hh)- Glioma-associated oncogene homolog (Gli) signaling regulates self-renewal of stem cells and implicates in a large number of human cancers. One of the Hh inhibitor, GDC-0449 is a potent small molecule inhibitor of Hedgehog-Gli pathway. It has been reported GDC-0449 showed high target specificity and demonstrated antiproliferative activity against tumors and it is now in clinical trial. Therefore, combination therapy using a BCR-ABL tyrosine kinase inhibitors and a Hedgehog-Gli inhibitor, GDC-0449 may help prevent CML relapse and these approaches may be expected to improve the outcomes of Ph-positive leukemia patients. In this study, we investigated the GDC-0449 efficacy by using the BCR-ABL positive cell lines, OM9;22, K562 and primary samples when leukemic cells were protected by the feeder cell line, S9 cells. We examined a comprehensive drug combination experiment using GDC-0449 and dual Src/ABL tyrosine kinase inhibitor, dasatinib. Gli proteins (Gli1, Gli2 and Gli3) were existed in Ph-positive cell lines. We found the cell numbers of OM9;22 were significantly increased with the feeder cell line, S9 cells compared to without S9 cells. The treatment of dasatinib exhibits cell growth inhibition partially against OM9;22 cells in the presence of feeder cell line, S9 cells. Caspase-3 activity by 100 nM dasatinib treatment was also reduced in the presence of S9 cells. 72 h of combined treatment of Ph-positive leukemia cells with 10 μM of GDC-0449 and 100 nM of dasatinib in the presence of feeder cell line, caused significantly more cytotoxicity than each drug alone. We next investigated the efficacy and intracellular signaling of GDC-0449. The treatment of GDC-0449 exhibits cell growth inhibition and induced apoptosis against OM9;22 cells in a dose and time dependent manner. Expression of Gli1 and Gli2 proteins were reduced after GDC-0449 treatment. 10 μM of GDC-0449 also inhibited the growth of Ph-positive primary samples by colony assay. Another Hh inhibitor, SANT-2 also exhibits cell growth inhibition against OM9;22 cells in a dose dependent manner. Data from this study suggested that administration of the Hh inhibitor, GDC-0449 may be a powerful strategy against Ph-positive leukemia cells and enhance cytotoxic effects of dasatinib in the presence of feeder cell. Disclosures: Ohyashiki: Nippon Shinyaku Co., Ltd.: Research Funding.
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Mian, Afsar Ali, Usva Zafar, Oliver Ottmann, Martin Ruthardt, and El-Nasir M. A. Lalani. "Activation of AKT/mTOR Pathway in Ph+ Acute Lymphoblastic Leukemia (ALL) Leads to Non-Mutational Resistance." Blood 134, Supplement_1 (November 13, 2019): 2570. http://dx.doi.org/10.1182/blood-2019-130898.
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Introduction: The t(9;22) (q34;q11) translocation results in the constative active BCR/ABL tyrosine kinase. Der22 involves the Breakpoint Cluster Region (BCR) gene locus with two principal breaks: a. M-bcr, encoding for the p210-BCR/ABL and b. m-bcr, encoding for the p185-BCR/ABL fusion proteins, respectively. BCR/ABL is the oncogenic driver of Chronic Myeloid Leukemia (CML) and 30% of adult Acute Lymphatic Leukemia (ALL). Activated BCR/ABL kinase is responsible for aberrant activation of multiple signaling pathways, such as JAK/STAT, PI3K/AKT and RAS/MAPK which eventually result in leukemic transformation. Successful targeting of BCR/ABL by selective tyrosine kinase inhibitors (TKIs) such as Imatinib, Nilotinib, Dasatinib and Ponatinib are used for the treatment of Philadelphia chromosome-positive (Ph+) leukemias. Most patients with CML in the early stage (CML-CP) treated with TKIs have increased overall survival. However, TKIs have not been as effective in patients with CML blast crisis (CML-BC) or Ph+ ALL. Point mutations in the tyrosine kinase domain (TKD) of BCR/ABL have emerged as the predominant cause of acquired resistance. These mutations are observed in up to 80% of patients with CML-BC and Ph+ ALL and in ~ 50% of Imatinib-resistant patients. In the remaining 20-50% of patients the mechanism of resistance to TKIs remains elusive. The aim of this study was to investigate the mechanism of non-mutational resistance in Ph+ ALL. Methods: As models for non-mutational resistance, we used patient derived long term cultures (PDLTCs) from Ph+ ALL patients with different levels of non-mutational drug resistance and the SupB15RT, a Ph+ ALL cell-line rendered resistant by exposure to increasing doses of Imatinib and cross-resistant against all approved ABL Kinase Inhibitors (AKIs). Cell proliferation was assessed by XTT/MTT and trypan blue dye exclusion. Signaling pathway proteins were assessed by Western Blot analysis. Chromosomal karyotyping was undertaken on single cell genomes using multi-color FISH (M-FISH) technology. Mutation analysis on the ABL kinase domain was done by sequencing the heminested PCR products obtained from SupB15-WT and SupB15RT cell-lines. Results: A non-mutational resistance cell line SupB15RT, was developed by exposing SupB15 cells to an increasing concentration of Imatinib over a 3 month period. SupB15RT were able to grow in 10 µM Imatinib. SupB15RT cells were karyotypically and mutationaly identical to SupB15 WT. All approved AKIs and allosteric inhibitors like GNF-2, ABL001 and Crizotinib were unable to inhibit growth of these cells, except for Dasatinib (IC50 40nM), a multi-target kinase inhibitor. Experiments to determine the mode of resistance revealed high level (3 fold) of activation of AKT/mTOR enabling these cells to grow and proliferate. We targeted the AKT/mTOR pathway using BKM-120 (PI3 Kinase inhibitor), BEZ-235 (PI3 Kinase and mTOR pathway) and Trorin1/Torin2 (mTORC1 and mTORC2) and found that Torin-1 and Torin-2 significantly inhibited proliferation of SupB15RT, in a dose dependent manner, with an IC50 of 11-20 nM. As Dasatinib alone inhibited growth of SupB15RT cells at 40-50nm concentrations, we combined Dasatinib with Torin1 and found that the combination of these two compounds had an additive inhibitory effect on cell growth. Following this we examined clinical samples from patients. We used three different Ph+ PDLTCs: a. HP (BCR/ABL negative), b. PH (BCR/ABL positive and responsive to TKIs) and c. BV (BCR/ABL positive and non-mutational resistant to TKIs). Interestingly, we found that AKT/mTOR pathway was activated in BV cells and its proliferation was inhibited by Torin1 with IC-50 of 50nM. Conclusion: Our experiments revealed an additional pathway involved in the evolution of non-mutational resistance in Ph+ ALL which could assist in developing novel targeted therapy for Ph+ ALL patient(s) with non-mutational resistance. Disclosures Ottmann: Celgene: Honoraria, Research Funding; Incyte: Honoraria, Research Funding; Amgen: Honoraria, Research Funding; Novartis: Honoraria; Takeda: Honoraria; Fusion Pharma: Honoraria; Pfizer: Honoraria; Roche: Honoraria.
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Zhang, Man, Xiaoyao Ma, Honglei Xu, Wenbo Wu, Xin He, Xiaoying Wang, Min Jiang, Yuanyuan Hou, and Gang Bai. "A natural AKT inhibitor swertiamarin targets AKT‐PH domain, inhibits downstream signaling, and alleviates inflammation." FEBS Journal 287, no. 9 (November 15, 2019): 1816–29. http://dx.doi.org/10.1111/febs.15112.
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Lee, Ming-Shyue, I.-Chu Tseng, Youhong Wang, Ken-ichi Kiyomiya, Michael D. Johnson, Robert B. Dickson, and Chen-Yong Lin. "Autoactivation of matriptase in vitro: requirement for biomembrane and LDL receptor domain." American Journal of Physiology-Cell Physiology 293, no. 1 (July 2007): C95—C105. http://dx.doi.org/10.1152/ajpcell.00611.2006.
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In live cells, autoactivation of matriptase, a membrane-bound serine protease, can be induced by lysophospholipids, androgens, and the polyanionic compound suramin. These structurally distinct chemicals induce different signaling pathways and cellular events that somehow, in a cell type-specific manner, lead to activation of matriptase immediately followed by inhibition of matriptase by hepatocyte growth factor activator inhibitor 1 (HAI-1). In the current study, we established an analogous matriptase autoactivation system in an in vitro cell-free setting and showed that a burst of matriptase activation and HAI-1-mediated inhibition spontaneously occurred in the insoluble fractions of cell homogenates and that this in vitro activation could be attenuated by a soluble suppressive factor(s) in cytosolic fractions. Immunofluorescence staining and subcellular fractionation studies revealed that matriptase activation occurred in the perinuclear regions. Solubilization of matriptase from cell homogenates by Triton X-100 or sonication of cell homogenates completely inhibited the effect, suggesting that matriptase activation requires proper lipid bilayer microenvironments, potentially allowing appropriate interactions of matriptase zymogens with HAI-1 and other components. Matriptase activation occurred in a narrow pH range (from pH 5.2 to 7.2), with a sharp increase in activation at the transition from pH 5.2 to 5.4, and could be completely suppressed by moderately increased ionic strength. Protease inhibitors only modestly affected activation, whereas 30 nM (5 μg/ml) of anti-matriptase LDL receptor domain 3 monoclonal antibodies completely blocked activation. These atypical biochemical features are consistent with a mechanism for autoactivation of matriptase that requires protein-protein interactions but not active proteases.
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Eide, Christopher A., Samantha L. Savage, Jade Bryant, Anupriya Agarwal, Daniel Bottomly, Beth Wilmot, Shannon K. McWeeney, et al. "Philadelphia Chromosome (Ph)-Positive Leukemia Patients Who Fail ABL1 Tyrosine Kinase Inhibitors Without BCR-ABL1 Kinase Domain Point Mutations Demonstrate Sensitivity To PI3-K/AKT Inhibitors." Blood 122, no. 21 (November 15, 2013): 3994. http://dx.doi.org/10.1182/blood.v122.21.3994.3994.
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Abstract Despite the impressive success of imatinib in chronic myeloid leukemia (CML), resistance to therapy remains an issue for approximately 15-20% of newly diagnosed chronic phase patients by 5 years and transient responses are the rule for patients with blast crisis CML or Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph+ ALL). The most frequent and well-characterized mechanism of resistance to imatinib is acquisition of point mutations in the BCR-ABL1 kinase domain which compromise drug binding. This form of resistance is largely well-handled by the newer, more potent ABL1 inhibitors nilotinib, dasatinib, and ponatinib. However, in the remainder of patients with resistance sustained inhibition of BCR-ABL1 kinase activity is necessary but no longer sufficient to inhibit cell growth, implicating activation of additional, thus far, poorly understood BCR-ABL1-independent mechanisms of growth and survival. In an effort to identify and validate novel pathways important in BCR-ABL-independent resistance to ABL1 kinase inhibitors, we screened a cohort of 30 patients with CML or Ph+ ALL exhibiting clinical resistance to at least one ABL1 kinase inhibitor without evidence of a drug-resistant BCR-ABL1 kinase domain mutation. Following informed consent, fresh primary mononuclear cells were isolated from each sample by Ficoll gradient centrifugation, plated ex vivo in the presence of a panel of clinical and pre-clinical small-molecule kinase inhibitors, and assessed for effects on viability after 3 days. Ex vivo resistance to ABL1 kinase inhibitors largely tracked with clinical resistance profiles, and considerable variation in sensitivity profiles for other inhibitors was observed. Among these data, we found a particularly interesting subset of patients whose cells demonstrated ex vivo sensitivity to one or more PI3-K/AKT inhibitors (n=6/30; 20%) including PI-103, BEZ235, CAL-101, and MK-2206. These findings are consistent with previous studies demonstrating PI3-K activation in select ABL1 kinase inhibitor-resistant CML cell lines (Quentmeier et al., J Hem Onc 2011) and in response to imatinib in pre-overt resistance (Burchert et al., Leukemia 2005). Among the effective PI3-K/AKT inhibitors, the most prominent were the dual class IA PI3-K/mTOR inhibitor PI-103 (IC50 range:38-110 nM) and BEZ235 (IC50 range: 20-210 nM). To further interrogate the mechanism(s) behind dependence on this pathway, samples of interest were analyzed by deep sequencing using a custom capture library encompassing ∼2000 kinases, phosphatases, and adaptor proteins. However, in contrast to previous reports of activating PIK3CA mutations in imatinib-resistant KCL-22 cells, we found PIK3CA to be wild-type in all samples. Despite a common sensitivity to PI3-K/AKT pathway inhibition, we found sequence variants were somewhat heterogeneous and direct kinase targets in this pathway were wild-type, suggesting other causative lesions which activate this pathway. Variants were prioritized based on known associations to this pathway, generated and evaluated in vitro for Ba/F3 cell transformation capacity, and validated for pathway activation and kinase inhibitor sensitivity. These results will be presented. Taken together, our findings suggest that a subset of patients with Ph+ leukemia who become refractory to ABL1 kinase inhibitors without a BCR-ABL1 kinase domain mutation demonstrate acquired dependence on the PI3-K/AKT axis, warranting further investigation of inhibitors of this pathway alone and in combination with ABL1 inhibitors as a molecularly targeted therapeutic strategy in patients. Disclosures: Off Label Use: Ruxolitinib - a JAK1/2 inhibitor that we propose can be used off-label for disease management of CSF3R-mutant neutrophilic leukemia. Deininger:BMS: Consultancy, Research Funding; ARIAD: advisory board, advisory board Other, Consultancy; Novartis: advisory board, advisory board Other, Consultancy, Research Funding; Celgene: Research Funding; Gilead: Research Funding. Tyner:Incyte Corporation: Research Funding. Druker:Novartis, Bristol-Myers Squibb, ARIAD & Incyte: Clin trial funding. OHSU holds contracts; Druker receives no salary/lab research funds. OHSU & Druker have financial interest in MolecularMD; technology used in some studies licensed to MolecularMD. This conflict has been reviewed and managed by OHSU. Other.
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DOWLER, Simon, Leire MONTALVO, Doreen CANTRELL, Nick MORRICE, and Dario R. ALESSI. "Phosphoinositide 3-kinase-dependent phosphorylation of the dual adaptor for phosphotyrosine and 3-phosphoinositides by the Src family of tyrosine kinase." Biochemical Journal 349, no. 2 (July 10, 2000): 605–10. http://dx.doi.org/10.1042/bj3490605.
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We recently identified a novel adaptor protein, termed dual adaptor for phosphotyrosine and 3-phosphoinositides (DAPP1), that possesses a Src homology (SH2) domain and a pleckstrin homology (PH) domain. DAPP1 exhibits a high-affinity interaction with PtdIns(3,4,5)P3 and PtdIns(3,4)P2, which bind to the PH domain. In the present study we show that when DAPP1 is expressed in HEK-293 cells, the agonists insulin, insulin-like growth factor-1 and epidermal growth factor induce the phosphorylation of DAPP1 at Tyr139. Treatment of cells with phosphoinositide 3-kinase (PI 3-kinase) inhibitors or expression of a dominant-negative PI 3-kinase prevent phosphorylation of DAPP1 at Tyr139, and a PH-domain mutant of DAPP1, which does not interact with PtdIns(3,4,5)P3 or PtdIns(3,4)P2, is not phosphorylated at Tyr139 following agonist stimulation of cells. Overexpression of a constitutively active form of PI 3-kinase induced the phosphorylation of DAPP1 in unstimulated cells. We demonstrated that Tyr139 of DAPP1 is likely to be phosphorylated in vivo by a Src-family tyrosine kinase, since the specific Src-family inhibitor, PP2, but not an inactive variant of this drug, PP3, prevented the agonist-induced tyrosine phosphorylation of DAPP1. Src, Lyn and Lck tyrosine kinases phosphorylate DAPP1 at Tyr139in vitro at similar rates in the presence or absence of PtdIns(3,4,5)P3, and overexpression of these kinases in HEK-293 cells induces the phosphorylation of Tyr139. These findings indicate that, following activation of PI 3-kinases, PtdIns(3,4,5)P3 or PtdIns(3,4)P2 bind to DAPP1, recruiting it to the plasma membrane where it becomes phosphorylated at Tyr139 by a Src-family tyrosine kinase.
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Cheng, Han, Tianji Chen, Merve Tor, Deborah Park, Qiyuan Zhou, Jason B. Huang, Nour Khatib, Lijun Rong, and Guofei Zhou. "A High-Throughput Screening Platform Targeting PDLIM5 for Pulmonary Hypertension." Journal of Biomolecular Screening 21, no. 4 (January 13, 2016): 333–41. http://dx.doi.org/10.1177/1087057115625924.
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Pulmonary arterial hypertension is a complex disease with multiple etiologic factors. PDLIM5, a member of the Enigma subfamily of PDZ and LIM domain protein family, contains an N-terminal PDZ domain and three LIM domains at its C-terminus. We have previously shown that overexpression of PDLIM5 prevents hypoxia-induced pulmonary hypertension (PH), and deletion of PDLIM5 in smooth muscle cells enhances hypoxia-induced PH in vivo. These results suggest that PDLIM5 may be a novel therapeutic target of PH. In this study, we aim to establish a high-throughput screening platform for PDLIM5-targeted drug discovery. We generated a stable mink lung epithelial cell line (MLEC) containing a transforming growth factor–β/Smad luciferase reporter with lentivirus-mediated suppression of PDLIM5 (MLEC-shPDLIM5) and measured levels of Smad2/3 and pSmad2/3. We found that in MLEC, suppression of PDLIM5 decreased Smad-dependent luciferase activity, Smad3, and pSmad3. We used MLEC-shPDLIM5 and a control cell line (MLEC-shCTL) to screen the Prestwick library (1200 compounds) and identified and validated paclitaxel as a PDLIM5 inhibitor in MLEC. Furthermore, we showed that paclitaxel inhibited Smad2 expression and Smad3 phosphorylation in A549 cells. Our study suggests that this system is robust and suitable for PDLIM5-targeted drug discovery.
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STRATFORD, Suzanne, Daryll B. DEWALD, and Scott A. SUMMERS. "Ceramide dissociates 3′-phosphoinositide production from pleckstrin homology domain translocation." Biochemical Journal 354, no. 2 (February 22, 2001): 359–68. http://dx.doi.org/10.1042/bj3540359.
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Numerous hormones, cytokines and transforming oncogenes activate phosphoinositide 3-kinase (PI-3K), a lipid kinase that initiates signal transduction cascades regulating cellular proliferation, survival, protein synthesis and glucose metabolism. PI-3K catalyses the production of the 3′-phosphoinositides PtdIns(3,4)P2 and PtdIns(3,4,5)P3, which recruit downstream effector enzymes to the membrane via their pleckstrin homology (PH) domains. Recent studies have indicated that another signalling lipid, the sphingolipid ceramide, inhibits several PI-3K-dependent events, including insulin-stimulated glucose uptake and growth-factor-stimulated cell survival. Here we show that ceramide analogues specifically prevent the recruitment of the PtdIns(3,4,5)P3-binding proteins Akt/protein kinase B (PKB) or the general receptor for phosphoinositides-1 (GRP1). Specifically, the short-chain ceramide derivative C2-ceramide inhibited the platelet-derived growth factor (PDGF)-stimulated translocation of full-length Akt/PKB, as well as truncated proteins encoding only the PH domains of Akt/PKB or GRP1. C2-ceramide did not alter the membrane localization of the PH domain for phospholipase Cδ, which preferentially binds PtdIns(4,5)P2, nor did it affect the PDGF-stimulated production of PtdIns(3,4)P2 or PtdIns(3,4,5)P3. Interestingly, a glucosylceramide synthase inhibitor, 1-phenyl-2-decanoylamino-3-morpholinopropan-1-ol (PDMP), shown previously to increase intracellular ceramide concentrations without affecting PI-3K [Rani, Abe, Chang, Rosenzweig, Saltiel, Radin and Shayman (1995) J. Biol. Chem. 270, 2859–2867], recapitulated the inhibitory effects of C2-ceramide on PDGF-stimulated Akt/PKB phosphorylation. These studies indicate that ceramide prevents the translocation of certain PtdIns(3,4,5)P3-binding proteins, despite the presence of a full complement of PtdIns(3,4)P2 or PtdIns(3,4,5)P3. Furthermore, these findings suggest a mechanism by which stimuli that induce ceramide synthesis could negate the fundamental signalling pathways initiated by PI-3K.
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Jiao, Yanting, Fukui Shen, Zhihua Wang, Lili Ye, Man Zhang, Jie Gao, Yuanyuan Hou, and Gang Bai. "Genipin, a natural AKT inhibitor, targets the PH domain to affect downstream signaling and alleviates inflammation." Biochemical Pharmacology 170 (December 2019): 113660. http://dx.doi.org/10.1016/j.bcp.2019.113660.
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KIMBER, Wendy A., Laura TRINKLE-MULCAHY, Peter C. F. CHEUNG, Maria DEAK, Louisa J. MARSDEN, Agnieszka KIELOCH, Stephen WATT, et al. "Evidence that the tandem-pleckstrin-homology-domain-containing protein TAPP1 interacts with Ptd(3,4)P2 and the multi-PDZ-domain-containing protein MUPP1 in vivo." Biochemical Journal 361, no. 3 (January 25, 2002): 525–36. http://dx.doi.org/10.1042/bj3610525.
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PtdIns(3,4,5)P3 is an established second messenger of growth-factor and insulin-induced signalling pathways. There is increasing evidence that one of the immediate breakdown products of PtdIns(3,4,5)P3, namely PtdIns(3,4)P2, whose levels are elevated by numerous extracellular agonists, might also function as a signalling molecule. Recently, we identified two related pleckstrin-homology (PH)-domain-containing proteins, termed ‘tandem-PH-domain-containing protein-1’ (TAPP1) and TAPP2, which interacted in vitro with high affinity with PtdIns(3,4)P2, but did not bind PtdIns(3,4,5)P3 or other phosphoinositides. In the present study we demonstrate that stimulation of Swiss 3T3 or 293 cells with agonists that stimulate PtdIns(3,4)P2 production results in the marked translocation of TAPP1 to the plasma membrane. This recruitment is dependent on a functional PtdIns(3,4)P2-binding PH domain and is inhibited by wortmannin, a phosphoinositide 3-kinase inhibitor that prevents PtdIns(3,4)P2 generation. A search for proteins that interact with TAPP1 identified the multi-PDZ-containing protein termed ‘MUPP1’, a protein possessing 13 PDZ domains and no other known modular or catalytic domains [PDZ is postsynaptic density protein (PSD-95)/Drosophila disc large tumour suppressor (dlg)/tight junction protein (ZO1)]. We demonstrate that immunoprecipitation of endogenously expressed TAPP1 from 293-cell lysates results in the co-immunoprecipitation of endogenous MUPP1, indicating that these proteins are likely to interact with each other physiologically. We show that TAPP1 and TAPP2 interact with the 10th and 13th PDZ domain of MUPP1 through their C-terminal amino acids. The results of the present study suggest that TAPP1 and TAPP2 could function in cells as adapter proteins to recruit MUPP1, or other proteins that they may interact with, to the plasma membrane in response to signals that elevate PtdIns(3,4)P2.
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Lorente-Rodríguez, Andrés, and Charles Barlowe. "Requirement for Golgi-localized PI(4)P in fusion of COPII vesicles with Golgi compartments." Molecular Biology of the Cell 22, no. 2 (January 15, 2011): 216–29. http://dx.doi.org/10.1091/mbc.e10-04-0317.
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The role of specific membrane lipids in transport between endoplasmic reticulum (ER) and Golgi compartments is poorly understood. Using cell-free assays that measure stages in ER-to-Golgi transport, we screened a variety of enzyme inhibitors, lipid-modifying enzymes, and lipid ligands to investigate requirements in yeast. The pleckstrin homology (PH) domain of human Fapp1, which binds phosphatidylinositol-4-phosphate (PI(4)P) specifically, was a strong and specific inhibitor of anterograde transport. Analysis of wild type and mutant PH domain proteins in addition to recombinant versions of the Sac1p phosphoinositide-phosphatase indicated that PI(4)P was required on Golgi membranes for fusion with coat protein complex II (COPII) vesicles. PI(4)P inhibition did not prevent vesicle tethering but significantly reduced formation of soluble n-ethylmaleimide sensitive factor adaptor protein receptor (SNARE) complexes between vesicle and Golgi SNARE proteins. Moreover, semi-intact cell membranes containing elevated levels of the ER-Golgi SNARE proteins and Sly1p were less sensitive to PI(4)P inhibitors. Finally, in vivo analyses of a pik1 mutant strain showed that inhibition of PI(4)P synthesis blocked anterograde transport from the ER to early Golgi compartments. Together, the data presented here indicate that PI(4)P is required for the SNARE-dependent fusion stage of COPII vesicles with the Golgi complex.
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Ding, Jie, Julia Romani, Margarete Zaborski, Roderick AF MacLeod, Stefan Nagel, Hans G. Drexler, and Hilmar Quentmeier. "Inhibition Of PI3K/mTOR Overcomes Nilotinib Resistance In BCR-ABL1 Positive Leukemia Cells." Blood 122, no. 21 (November 15, 2013): 4906. http://dx.doi.org/10.1182/blood.v122.21.4906.4906.
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Abstract Expression of the Philadelphia chromosome (Ph), i.e., the t(9;22) chromosomal translocation and the formation of the BCR-ABL1 fusion protein, is the hallmark of chronic myeloid leukemia (CML). Tyrosine kinase inhibitors (TKI), such as imatinib and nilotinib, have emerged as leading compounds to treat CML. Translocation t(9;22) does not only occur in CML, 20-30% of acute lymphoblastic leukemia (ALL) are also found to carry the Ph. However, TKIs are not as effective in the treatment of Ph+ ALL as in CML. In this study, the Ph+ cell lines JURL-MK2 and SUP-B15 were used to investigate TKI resistance mechanisms and sensitization of Ph+ tumor cells to TKI treatment. The annexin V/PI (propidium iodide) assay revealed that nilotinib induced apoptosis in JURL-MK2 cells, but not in SUP-B15 cells. There was no mutation in the tyrosine kinase domain of BCR-ABL1 in both cell lines. SUP-B15 cells were not generally irresponsive to TKI, evidenced by dephosphorylation of the BCR-ABL1 downstream target GAB2 (Grb-associated binder-2). The resistance to apoptosis after nilotinib treatment was accompanied by the constitutive and nilotinib irresponsive activation of the phosphoinositide 3-kinase (PI3K) pathway. Treatment of SUP-B15 with the dual PI3K/mammalian target of rapamycin (mTOR) inhibitor BEZ235 alone induced apoptosis in a low percentage of cells, combination of nilotinib and BEZ235 led to a synergistic effect. Caspase 3 and PARP (poly ADP ribose polymerase) cleavage confirmed that apoptosis was induced after combined treatment. The main role of PI3K/mTOR inhibitor BEZ235 and the reason for apoptosis in the nilotinib-resistant cells was the block of the translational machinery, leading to the rapid down-regulation of anti-apoptotic protein MDM2 (human homolog of the murine double minute 2, hMDM2). The current findings suggest that MDM2 may be a therapeutic target to increase TKI-mediated apoptosis and that the combination of PI3K/mTOR dual inhibitor and TKI inhibitor might turn out to be a novel strategy for TKI-resistant BCR-ABL1 positive leukemia. Disclosures: No relevant conflicts of interest to declare.
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Saric, Arman, Lars zur Brügge, Dirk Müller-Pompalla, Thomas Rysiok, Solenne Ousson, Bruno Permanne, Anna Quattropani, Michael Busch, Dirk Beher, and Ishrut Hussain. "Development and Characterization of a Novel Membrane Assay for Full-Length BACE-1 at pH 6.0." Journal of Biomolecular Screening 18, no. 3 (September 27, 2012): 277–85. http://dx.doi.org/10.1177/1087057112462237.
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β-Site amyloid precursor protein cleaving enzyme–1 (BACE-1) is a transmembrane aspartic protease that mediates the initial cleavage of the amyloid precursor protein (APP), leading to the generation of amyloid-β (Aβ) peptides that are thought to be causative of Alzheimer’s disease (AD). Consequently, inhibition of BACE-1 is an attractive therapeutic approach for the treatment of AD. In general, in vitro biochemical assays to monitor BACE-1 activity have used the extracellular domain of the protein that contains the catalytic active site. This form of BACE-1 is catalytically active at acidic pH and cleaves APP-based peptide substrates at the β-site. However, this form of BACE-1 does not mimic the natural physiology of BACE-1 and shows minimal activity at pH 6.0, which is more representative of the pH within the intracellular compartments where BACE-1 resides. Moreover, high-throughput screens with recombinant BACE-1 at pH 4.5 have failed to identify tractable leads for drug discovery, and hence, BACE-1 inhibitor development has adopted a rational drug design approach. Here we describe the development and validation of a novel membrane assay comprising full-length BACE-1 with measurable activity at pH 6.0, which could be used for the identification of novel inhibitors of BACE-1.
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Prabhakara, Chaitra, Rashmi Godbole, Parijat Sil, Sowmya Jahnavi, Shah-e.-Jahan Gulzar, Thomas S. van Zanten, Dhruv Sheth, et al. "Strategies to target SARS-CoV-2 entry and infection using dual mechanisms of inhibition by acidification inhibitors." PLOS Pathogens 17, no. 7 (July 12, 2021): e1009706. http://dx.doi.org/10.1371/journal.ppat.1009706.
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Many viruses utilize the host endo-lysosomal network for infection. Tracing the endocytic itinerary of SARS-CoV-2 can provide insights into viral trafficking and aid in designing new therapeutic strategies. Here, we demonstrate that the receptor binding domain (RBD) of SARS-CoV-2 spike protein is internalized via the pH-dependent CLIC/GEEC (CG) endocytic pathway in human gastric-adenocarcinoma (AGS) cells expressing undetectable levels of ACE2. Ectopic expression of ACE2 (AGS-ACE2) results in RBD traffic via both CG and clathrin-mediated endocytosis. Endosomal acidification inhibitors like BafilomycinA1 and NH4Cl, which inhibit the CG pathway, reduce the uptake of RBD and impede Spike-pseudoviral infection in both AGS and AGS-ACE2 cells. The inhibition by BafilomycinA1 was found to be distinct from Chloroquine which neither affects RBD uptake nor alters endosomal pH, yet attenuates Spike-pseudovirus entry. By screening a subset of FDA-approved inhibitors for functionality similar to BafilomycinA1, we identified Niclosamide as a SARS-CoV-2 entry inhibitor. Further validation using a clinical isolate of SARS-CoV-2 in AGS-ACE2 and Vero cells confirmed its antiviral effect. We propose that Niclosamide, and other drugs which neutralize endosomal pH as well as inhibit the endocytic uptake, could provide broader applicability in subverting infection of viruses entering host cells via a pH-dependent endocytic pathway.
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Nakagawa, Ryo, Yoriko Saito, Shinsuke Takagi, Sadaaki Takata, Hanae Amitani, Mikiko Endo, Naoyuki Uchida, et al. "Identification of Patient-Specific Anti-Apoptotic Molecules As Therapeutic Targets in Poor Prognosis Acute Lymphoblastic Leukemia (ALL)." Blood 138, Supplement 1 (November 5, 2021): 1236. http://dx.doi.org/10.1182/blood-2021-147625.
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Abstract Relapse and refractory ALL shows dismal prognosis despite recent progress in intensive chemotherapy and development of molecular targeting agents. In this study, we aimed to identify vulnerabilities in genetically-diverse ALL and to find additional therapeutic targets in Philadelphia chromosome-positive (Ph+) ALL and chronic myeloid myeloid leukemia (CML) to overcome BCR-ABL tyrosine kinase inhibitor (TKI) resistance. To this end, we performed in vitro drug screening using 23 human ALL samples (9 Ph+ ALL, 5 MLL-ALL, 1 ALL with ETV-RUNX1, 5 B-ALL, not otherwise specified and 4 T-ALL), 10 CML samples and eight mixed phenotype acute leukemia (MPAL) samples (four B-Myeloid and four T-Myeloid). Based on our recent findings in AML (Hashimoto, Saito et al., Nature Cancer 2021), we treated leukemia-engrafting cells with small molecules targeting anti-apoptotic molecules (BCL-2, MCL-1 or BIRC) or molecules associated with cell division (AURKB) in addition to tyrosine kinase inhibitors. Among these compounds, BIRC inhibitor and venetoclax exhibited great efficacy. Responsiveness to each compound was: 28 of 41 for BIRC inhibitor (68.3%), 24 of 40 for venetoclax (60%), 11 of 37 for MCL-1 inhibitor (29.7%) and 4 of 37 for AURKB inhibitor (10.8%). We found differential sensitivity between T-ALL/MPAL T-myeloid and CML. Seven out of eight T-ALL and MPAL T-myeloid samples were highly sensitive to venetoclax (87.5%), while nine out of 10 CML samples were responsive to BIRC inhibition (90%). On the other hand, among Ph+ ALL/MPAL and Ph- B-ALL/MPAL B-myeloid samples, sensitivities to BIRC inhibitor and venetoclax were variable. To identify determinants of sensitivity to compounds, we examined the relation between mutational profile and in vitro leukemia elimination through targeted DNA sequencing for 79 lymphoid and myeloid malignancy-associated somatic mutations. Consistent with our previous study, CBL-mutated leukemia showed higher sensitivity to BIRC inhibitor (four of five cases) compared with venetoclax (two of five cases). Among genes related to RAS signaling pathway, KRAS mutations were most frequent (n=6). While five of six KRAS-mutated cases were BIRC inhibitor sensitive (83.3%), three of six cases were sensitive to venetoclax (50%). For cases with mutations in BCR-ABL1 kinase domain, the sensitivity to BIRC inhibitor and venetoclax was variable. Finally, we went on to setup in vivo experiments to elucidate if targeting the patient-specific vulnerabilities resulted in potent therapeutic efficacy against patient leukemic cells. We created patient-derived xenograft (PDX) models of 5 Ph+ ALL/MPAL, 2 Ph- B-ALL/MPAL, 2 T-ALL/MPAL and 2 CML cases. For Ph+ ALL/MPAL and CML, BIRC inhibitor and/or venetoclax combined with dexamethasone (DEX) and TKI achieved effective in vivo elimination of leukemic cells as predicted by in vitro experiments. Combination therapy showed almost complete elimination of Ph+ leukemic cells even in the presence of T315I mutation. For Ph- B-ALL/MPAL and T-ALL/MPAL, preliminary in vivo experiments showed additional inhibition of BIRC and BCL-2 resulted in more profound reduction of leukemic cells in BM compared with DEX alone (Combination: 2.1% [0.25-8] of hCD45+ leukemic cells, n=13 vs. DEX alone: 67.2% [59-76.5], n=11, median [IQR], p<0.001). We found targeting anti-apoptotic molecules in combination with DEX and/or TKI eradicated human genetically-diverse ALL, MPAL and CML cells both in vitro and in vivo. Inhibition of BIRC and BCL-2 overcame glucocorticoid resistance of Ph- ALL. Altogether, our results may offer precision medicine approach and contribute to improvement of clinical outcome in treatment-resistant ALL, CML and MPAL. Disclosures Uchida: Sumitomo Dainippon Pharma Co., Ltd.: Honoraria; Otsuka Pharmaceutical Co., Ltd.: Honoraria; Astellas Pharma Inc.: Honoraria; Chugai Pharmaceutical Co., Ltd.: Honoraria; Novartis Pharma Inc.: Honoraria. Harigae: Ono pharma: Honoraria, Other: Subsidies or Donations; Astellas Pharma: Other: Subsidies or Donations; Kyowakirin: Other: Subsidies or Donations; Janssen Pharma: Honoraria; Chugai Pharma: Honoraria; Novartis Pharma: Honoraria, Research Funding; Bristol Myers Squibb: Honoraria.
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Linev, Alexandar J., Hristo J. Ivanov, Ivan G. Zhelyazkov, Hristina Ivanova, Veselina S. Goranova-Marinova, and Vili K. Stoyanova. "Mutations Associated with Imatinib Mesylate Resistance - Review." Folia Medica 60, no. 4 (December 1, 2018): 617–23. http://dx.doi.org/10.2478/folmed-2018-0030.
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Abstract Chronic myeloid leukemia (CML) arises from the fusion of the BCR and the ABL1 genes. The BCR gene (chromosome 22q11.2) and the ABL1 gene (chromosome 9q34) fuse together due to reciprocal chromosome translocation forming the Philadelphia chromosome (Ph). This fusion gene codes tyrosine kinase which accelerates the cell division and reduces DNA repair. Imatinib mesylate is a selective inhibitor of this tyrosine kinase. It is the first-line treatment for CML-patients. However, it became clear that Philadelphia-positive (Ph+) cells could evolve to elude inhibition due to point mutations within the BCR-ABL kinase domain. To date more than 40 mutations have been identified and their early detection is important for clinical treatment. With the development of the new tyrosine kinase inhibitors (TKIs), associated with these mutations, the resistance problem seems to diminish, as some of the new drugs are less prone to resistance. The aim of this review is to focus on the diff erent mutations leading to resistance.
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Howe, Nicole, Mariangela Ceruso, Edward Spink, and J. Paul G. Malthouse. "pH stability of the stromelysin-1 catalytic domain and its mechanism of interaction with a glyoxal inhibitor." Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics 1814, no. 10 (October 2011): 1394–403. http://dx.doi.org/10.1016/j.bbapap.2011.07.004.
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Roth, Christian, Olga V. Moroz, Johan P. Turkenburg, Elena Blagova, Jitka Waterman, Antonio Ariza, Li Ming, et al. "Structural and Functional Characterization of Three Novel Fungal Amylases with Enhanced Stability and pH Tolerance." International Journal of Molecular Sciences 20, no. 19 (October 3, 2019): 4902. http://dx.doi.org/10.3390/ijms20194902.
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Amylases are probably the best studied glycoside hydrolases and have a huge biotechnological value for industrial processes on starch. Multiple amylases from fungi and microbes are currently in use. Whereas bacterial amylases are well suited for many industrial processes due to their high stability, fungal amylases are recognized as safe and are preferred in the food industry, although they lack the pH tolerance and stability of their bacterial counterparts. Here, we describe three amylases, two of which have a broad pH spectrum extending to pH 8 and higher stability well suited for a broad set of industrial applications. These enzymes have the characteristic GH13 α-amylase fold with a central (β/α)8-domain, an insertion domain with the canonical calcium binding site and a C-terminal β-sandwich domain. The active site was identified based on the binding of the inhibitor acarbose in form of a transglycosylation product, in the amylases from Thamnidium elegans and Cordyceps farinosa. The three amylases have shortened loops flanking the nonreducing end of the substrate binding cleft, creating a more open crevice. Moreover, a potential novel binding site in the C-terminal domain of the Cordyceps enzyme was identified, which might be part of a starch interaction site. In addition, Cordyceps farinosa amylase presented a successful example of using the microseed matrix screening technique to significantly speed-up crystallization.
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Najafov, Ayaz, Eeva M. Sommer, Jeffrey M. Axten, M. Phillip Deyoung, and Dario R. Alessi. "Characterization of GSK2334470, a novel and highly specific inhibitor of PDK1." Biochemical Journal 433, no. 2 (December 22, 2010): 357–69. http://dx.doi.org/10.1042/bj20101732.
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PDK1 (3-phosphoinositide-dependent protein kinase 1) activates a group of protein kinases belonging to the AGC [PKA (protein kinase A)/PKG (protein kinase G)/PKC (protein kinase C)]-kinase family that play important roles in mediating diverse biological processes. Many cancer-driving mutations induce activation of PDK1 targets including Akt, S6K (p70 ribosomal S6 kinase) and SGK (serum- and glucocorticoid-induced protein kinase). In the present paper, we describe the small molecule GSK2334470, which inhibits PDK1 with an IC50 of ~10 nM, but does not suppress the activity of 93 other protein kinases including 13 AGC-kinases most related to PDK1 at 500-fold higher concentrations. Addition of GSK2334470 to HEK (human embryonic kidney)-293, U87 or MEF (mouse embryonic fibroblast) cells ablated T-loop residue phosphorylation and activation of SGK isoforms and S6K1 induced by serum or IGF1 (insulin-like growth factor 1). GSK2334470 also inhibited T-loop phosphorylation and activation of Akt, but was more efficient at inhibiting Akt in response to stimuli such as serum that activated the PI3K (phosphoinositide 3-kinase) pathway weakly. GSK2334470 inhibited activation of an Akt1 mutant lacking the PH domain (pleckstrin homology domain) more potently than full-length Akt1, suggesting that GSK2334470 is more effective at inhibiting PDK1 substrates that are activated in the cytosol rather than at the plasma membrane. Consistent with this, GSK2334470 inhibited Akt activation in knock-in embryonic stem cells expressing a mutant of PDK1 that is unable to interact with phosphoinositides more potently than in wild-type cells. GSK2334470 also suppressed T-loop phosphorylation and activation of RSK2 (p90 ribosomal S6 kinase 2), another PDK1 target activated by the ERK (extracellular-signal-regulated kinase) pathway. However, prolonged treatment of cells with inhibitor was required to observe inhibition of RSK2, indicating that PDK1 substrates possess distinct T-loop dephosphorylation kinetics. Our data define how PDK1 inhibitors affect AGC signalling pathways and suggest that GSK2334470 will be a useful tool for delineating the roles of PDK1 in biological processes.
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Minami, Miho, Yosuke Minami, Yachiyo Kuwatsuka, Hitoshi Kiyoi, and Tomoki Naoe. "Biomarkers In Cell Death of Imatinib-Resistant Ph-Leukemia Cells During Treatment with mTOR Inhibitor, Everolimus." Blood 116, no. 21 (November 19, 2010): 3988. http://dx.doi.org/10.1182/blood.v116.21.3988.3988.
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Abstract Abstract 3988 Aberrant activation of mammalian target of rapamycin (mTOR) signaling pathway has been reported in hematological malignancies including leukemia initiating cells. Although rapamycin and its analogs have proven effective as anticancer agents, the mechanism of action and the solid biomarkers of response have not been fully elucidated. We investigated detailed biomarkers during the cell death of imatinib (IM)-resistant Ph-positive (Ph+) leukemia cells due to quiescence or mutations at the ABL-kinase domain after treatment with mTOR inhibitor, everolimus (Eve, RAD001). Ph+ leukemic NOD/SCID/IL2rγnull (NOG) mice cells were long co-cultured with S17 stromal cells and treated with IM and Eve. While slow-cycling (Hoechst 33342low/Pyronin Ylow) CD34+ cells were insensitive to IM in spite of BCR-ABL-dephosphorylation, combination treatment with IM and Eve induced substantial cell death including the CD34+ population. In Baf3/p210T315I cells, IM-resistant Ph+ leukemia cell line harboring T315I-mutation, Eve also induced cell death with low IC50 values in PI-exclusion assays. In murine model cutaneously injected with Baf3/p210T315I cells, in vivo-treatment with Eve decreased tumor formation. In these systems during treatment with Eve, we did not observe evident dephosphorylations of BCR-ABL, mTOR itself and 4EBP1, but rapid S6K-dephosphorylation with lower doses and decreased expression of MCL-1. Furthermore, the feedback-loop effects such as reversely increased phosphorylations of AKT (Ser473) and FOXO1/3a were also detected during the cell death. We are now investigating more efficient strategies using inhibitors screening kit and also planning to examine new generation of mTOR inhibitors to overcome the IM-resistance due to quiescence or T315I-mutation. Disclosures: Naoe: Kyowa-Kirin: Research Funding; Novartis: Research Funding; Janssen: Research Funding.
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Firestein, Ron, and Michael L. Cleary. "Pseudo-phosphatase Sbf1 contains an N-terminal GEF homology domain that modulates its growth regulatory properties." Journal of Cell Science 114, no. 16 (August 15, 2001): 2921–27. http://dx.doi.org/10.1242/jcs.114.16.2921.
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Sbf1 (SET binding factor 1) is a pseudo-phosphatase related to the myotubularin family of dual specificity phosphatases, some of which have been implicated in cellular growth and differentiation by virtue of their mutation in human genetic disorders. Sbf1 contains germline-encoded alterations of its myotubularin homology domain that render it non-functional as a phosphatase. We report here the complete structure of Sbf1 and further characterization of its growth regulatory properties. In addition to its similarity to myotubularin, the predicted full-length Sbf1 protein contains pleckstrin (PH)and GEF homology domains that are conserved in several proteins implicated in signaling and growth control. Forced expression of wild-type Sbf1 in NIH 3T3 cells inhibited their proliferation and altered their morphology. These effects required intact PH, GEF and myotubularin homology domains, implying that growth inhibition may be an intrinsic property of wild-type Sbf1. Conversely, deletion of its conserved N-terminal 44 amino acids alone was sufficient to convert Sbf1 from an inhibitor of cellular growth to a transforming protein in NIH 3T3 cells. Oncogenic forms of Sbf1 partially localized to the nucleus, in contrast to the exclusively cytoplasmic subcellular localization of endogenous Sbf1 in all cell lines and mammalian tissues tested. These data show that the N-terminal GEF homology domain serves to inhibit the transforming effects of Sbf1, possibly sequestering the protein to the cytoplasm, and suggest that this region may be a modulatory domain that relays growth control signals.
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Boedtkjer, Ebbe, and Christian Aalkjaer. "Insulin inhibits Na+/H+ exchange in vascular smooth muscle and endothelial cells in situ: involvement of H2O2 and tyrosine phosphatase SHP-2." American Journal of Physiology-Heart and Circulatory Physiology 296, no. 2 (February 2009): H247—H255. http://dx.doi.org/10.1152/ajpheart.00725.2008.
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Insulin signals through several intracellular pathways. Here, we tested the hypothesis that insulin modulates Na+/H+ exchange (NHE) activity in vascular cells through H2O2-mediated inhibition of tyrosine phosphatase Src homology 2 domain containing tyrosine phosphatase 2 (SHP-2). We measured intracellular pH (pHi) in isolated mouse mesenteric arteries using fluorescence confocal and wide-field microscopy. In the absence of CO2/HCO3−, removal of bath Na+ produced endothelial acidification (ΔpHi = −0.71 ± 0.12) inhibited by cariporide. Cariporide reduced endothelial steady-state pHi (ΔpHi=−0.28 ± 0.08). Insulin and H2O2 acidified endothelial cells 0.2–0.3 pH units and reduced the acidification upon Na+ removal by ∼65%. Cariporide abolished the effect of insulin and H2O2. In vascular smooth muscle cells, H2O2 produced intracellular acidification (ΔpHi = −0.48 ± 0.06) as did high concentrations of insulin (ΔpHi = −0.03 ± 0.01). NHE activity after an NH4+ prepulse was ∼80% attenuated by H2O2 and ∼40% by high insulin concentrations. H2O2 had no effect on Na+-HCO3− cotransport activity. NHE1 (slc9a1) was the only plasma membrane NHE isoform detected in mouse mesenteric arteries by RT-PCR analyses. In both cell types, polyethylene glycol catalase abolished the effect of insulin on pHi. Exposure to insulin increased the intracellular concentration of reactive oxygen species estimated with the fluorophore 5-(6)-chloromethyl-2′,7′-dichlorodihydrofluorescein. The SHP-2 selective inhibitor NSC-87877 and protein tyrosine phosphatase (PTP) inhibitor IV reduced steady-state pHi up to 0.3 pH units and inhibited NHE activity 60–80%; when applied in combination with insulin or H2O2, no further effect was obtained. We conclude that NHE contributes to pHi regulation in arterial endothelial and smooth muscle cells in situ and is inhibited by insulin and H2O2. We propose that insulin signaling involves H2O2 and inhibition of PTP SHP-2.
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EBLE, Johannes A., and Danny S. TUCKWELL. "The α2β1 integrin inhibitor rhodocetin binds to the A-domain of the integrin α2 subunit proximal to the collagen-binding site." Biochemical Journal 376, no. 1 (November 15, 2003): 77–85. http://dx.doi.org/10.1042/bj20030373.
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Rhodocetin is a snake venom protein that binds to α2β1 integrin, inhibiting its interaction with its endogenous ligand collagen. We have determined the mechanism by which rhodocetin inhibits the function of α2β1. The interaction of α2β1 with collagen and rhodocetin differed: Ca2+ ions and slightly acidic pH values increased the binding of α2β1 integrin to rhodocetin in contrast with their attenuating effect on collagen binding, suggesting that rhodocetin preferentially binds to a less active conformation of α2β1 integrin. The α2A-domain [von Willebrand factor domain A homology domain (A-domain) of the integrin α2 subunit] is the major site for collagen binding to α2β1. Recombinant α2A-domain bound rhodocetin, demonstrating that the A-domain is also the rhodocetin-binding domain. Although the interaction of α2β1 with rhodocetin is affected by altering divalent cations, the interaction of the A-domain was divalent-cation-independent. The rhodocetin-binding site on the α2A-domain was mapped first by identifying an anti-α2 antibody that blocked rhodocetin binding and then mapping the epitope of the antibody using human–mouse α2A-domain chimaeras; and secondly, by binding studies with α2A-domain, which bear point mutations in the vicinity of the mapped epitope. In this way, the rhodocetin-binding site was identified as the α3–α4 loop plus adjacent α-helices. This region is known to form part of the collagen-binding site, thus attaining a mainly competitive mode of inhibition by rhodocetin.
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Hantschel, Oliver, Sina Reckel, Hamelin Romain, Florence Armand, Delphine Harduin, Shohei Koide, Volker Dötsch, and Marc Moniatte. "Comprehensive Analysis of the Structural, Biochemical and Signaling Differences of the p210 and p185 Isoforms of Bcr-Abl in CML and B-ALL." Blood 128, no. 22 (December 2, 2016): 4238. http://dx.doi.org/10.1182/blood.v128.22.4238.4238.
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Abstract Distinct chromosomal translocation breakpoints result in the expression of two major isoforms of Bcr-Abl - p210 and p185 - from the Philadelphia (Ph)-chromosome. While p210 is the molecular hallmark of Chronic Myeloid Leukemia (CML), in Ph-positive B-cell Acute Lymphoblastic Leukemia (B-ALL), ~1/3 of the patients express p210 and ~2/3 express p185. In mouse models, under certain experimental conditions, expression of p185 causes disease with a shorter latency and more B-ALL than CML-like leukemias as did p210. Ph-positive B-ALL patients show a poorer response and frequent resistance to tyrosine kinase inhibitor (TKI) therapy as compared to CML patients. Specific intrinsic differences in p185 and p210 Bcr-Abl signaling have long been hypothesized, but have never been studied in a comparative, unbiased and comprehensive manner. We have now: (i) Mapped the differences in the p210 and p185 protein interaction network: We performed an unbiased proteomics analysis of the composition of the endogenous p210 and p185 protein complexes in murine cell lines using SILAC (stable isotope labeling with amino acids in cell culture) quantification. The interactome analysis confirmed previously reported p210 interactors in human CML cells and identified unexpectedly many interactors that preferentially associate with either p185 or p210, including an endocytosis-regulating complex (enriched in p185) and a phosphatase complex (enriched in p210). Several differential interactors were validated in a panel of nine human CML and B-ALL cell lines. (ii) Performed a comprehensive phospho-proteomics study in p210 and p185 cell lines: Using phospho-tyrosine, as well as general phospho-peptide enrichment strategies coupled to SILAC quantification, we mapped >800 phosphorylation sites and identified differentially activated signaling by p210 and p185, including differential activation of the STAT5 and STAT3 transcription factors, SRC kinases and ERK1/2 signaling. These findings were validated in a panel of nine human CML and B-ALL cell lines. (iii) Determined the atomic structures of the Dbl-homology (DH) and pleckstrin homology (PH) domains of p210: The DH-PH domain unit of p210 is the only structural difference to p185 and has remained the only structurally uncharacterized region of Bcr-Abl. We solved different structures of these domains by NMR spectroscopy, X-ray crystallography, as well as Small-Angle X-ray scattering, alone and in complex with an engineered monobody inhibitor targeting the DH domain. While the DH-PH structural motif is found in Rho guanine nucleotide exchange factors (Rho GEF), our results have not provided evidence that the p210 DH-PH domain acts as a Rho GEF or binds Rho GTPases, in contrast to previous reports. On the other hand, we found the PH domain to be important for specific membrane binding of p210 and we determined the binding specificity of the PH domain to certain phospho-inositide lipids. Our results provide the first systematic insight showing critical differences in protein-protein interactions, signaling and structure of p210 and p185 Bcr-Abl. This will contribute to a better understanding of Bcr-Abl dependent signaling, mechanisms of oncogenic transformation, resulting disease pathobiology and responses to TKIs. Disclosures Hantschel: Ariad Pharmaceuticals: Honoraria.
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de Hoog, Carmen L., Wing-Tze Fan, Marni D. Goldstein, Michael F. Moran, and C. Anne Koch. "Calmodulin-Independent Coordination of Ras and Extracellular Signal-Regulated Kinase Activation by Ras-GRF2." Molecular and Cellular Biology 20, no. 8 (April 15, 2000): 2727–33. http://dx.doi.org/10.1128/mcb.20.8.2727-2733.2000.
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ABSTRACT Ras-GRF2 (GRF2) is a widely expressed, calcium-activated regulator of the small-type GTPases Ras and Rac. It is a multidomain protein composed of several recognizable sequence motifs in the following order (NH2 to COOH): pleckstrin homology (PH), coiled-coil, ilimaquinone (IQ), Dbl homology (DH), PH, REM (Ras exchanger motif), PEST/destruction box, Cdc25. The DH and Cdc25 domains possess guanine nucleotide exchange factor (GEF) activity and interact with Rac and Ras, respectively. The REM-Cdc25 region was found to be sufficient for maximal activation of Ras in vitro and in vivo caused Ras and extracellular signal-regulated kinase (ERK) activation independent of calcium signals, suggesting that, at least when expressed ectopically, it contains all of the determinants required to access and activate Ras signaling. Additional mutational analysis of GRF2 indicated that the carboxyl PH domain imparts a modest inhibitory effect on Ras GEF activity and probably normally participates in intermolecular interactions. A variant of GRF2 missing the Cdc25 domain did not activate Ras and functions as an inhibitor of wild-type GRF2, presumably by competing for interactions with molecules other than calmodulin, Ras, and ligands of the PH domain. The binding of calmodulin was found to require several amino-terminal domains of GRF2 in addition to the IQ sequence, and no correlation between calmodulin binding by GRF2 and its ability to directly activate Ras and indirectly stimulate the mitogen-activated protein (MAP) kinase ERK in response to calcium was found. The precise role of the GRF2-calmodulin association, therefore, remains to be determined. A GRF2 mutant missing the IQ sequence was competent for Ras activation but failed to couple this to stimulation of the ERK pathway. This demonstrates that Ras-GTP formation is not sufficient for MAP kinase signaling. We conclude that in addition to directly activating Ras, GRF2, and likely other GEFs, promote the assembly of a protein network able to couple the GTPase with particular effectors.
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Mian, Afsar Ali, Hadiqa Raees, Sujjawal Ahmad, Oliver Ottmann, and El-Nasir M. A. Lalani. "Arsenic Trioxide Suppresses Growth of BCR-ABL1 Positive Cells with "Gatekeeper" or Compound Mutation." Blood 138, Supplement 1 (November 5, 2021): 4346. http://dx.doi.org/10.1182/blood-2021-154511.
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Abstract Introduction: Chronic myeloid leukemia (CML) and 30% of adult acute lymphatic leukemia (ALL) are characterized by the Philadelphia chromosome (Ph +), having a (9;22) chromosomal translocation. The BCR-ABL1 fusion protein is the hallmark of Ph + leukemia. BCR-ABL1 is characterized by constitutively activated ABL1 tyrosine kinase activity that determines its transformation potential. Tyrosine kinase inhibitors (TKI) have greatly improved the overall prognosis of these diseases. However, unsatisfactory responses in advanced disease stages, resistance and long-term tolerability of BCR-ABL1 inhibitors represent major clinical problems. The most important resistance mechanism against TKIs is the acquisition of point mutations within the BCR-ABL1 kinase domain that impair drug binding, restoring the oncoprotein's constitutively active tyrosine kinase activity. The selection of leukemic clones driven by BCR-ABL1 harboring point mutations, such as the E255K, Y253F/H (P-loop), H396R (activation loop) or the T315I (gatekeeper). Second- and third generation TKIs such as nilotinib, dasatinib, and ponatinib effectively overcome point mutation-mediated resistance. Ponatinib is the only U.S. Food and Drug Administration approved TKI with activity against all known BCR-ABL1 point mutations, including BCR-ABL1-T315I. However, the emergence of compound mutations (two mutations within the same BCR-ABL1 allele) has been linked to resistance to all approved TKIs, including ponatinib, posing a clinical challenge with limited treatment options. The anti-cancer agent arsenic trioxide (ATO) has been used to treat patients with acute promyelocytic leukemia (APL). APL patients respond very well to ATO therapy and achieve complete remission, possibly through induction of apoptosis and differentiation. In addition, it has been demonstrated that combined treatment of ATO with interferon or nilotinib significantly suppressed cell proliferation. However, the potential effects of ATO on BCR-ABL1 mutations and especially on compound mutation is not apparent. This study aimed to investigate the role of ATO in BCR-ABL1 resistant mutations, including compound mutation in Ph + leukemias. Methods: We undertook preclinical evaluation of ATO and compared it with approved TKIs e.g. imatinib, nilotinib, dasatinib, ponatinib and ABL inhibitor asciminib, in vitro models of CML and primary patient-derived long term cultures (PD-LTC) of Ph + ALL patients with or without mutation. The effects on mutational resistance were investigated in Ba/F3 cells expressing BCR-ABL1 with T315I mutation and T315I-E255K mutation. For non-mutational resistance, we used PD-LTCs from Ph + ALL patients with different levels of non-mutational drug resistance. Cell proliferation was assessed by XTT. Results: ATO efficiently inhibited the growth of all PD-LTCs in cellular assays at dosages of 200-500nM. It also suppressed the growth of Ph + PD-LTC with non- mutational resistance (BV) and the BCR-ABL1-T315I positive PD-LTC (KO) in this dosage range. In all modelsWe treated Ba/F3 cells expressing native BCR-ABL1, BCR-ABL1-T315I mutation and BCR-ABL1-T315I-E255K (compound mutation) with increasing concentrations of imatinib (250, 500 and 1000nM), nilotinib (100, 200 and 400nM), dasatinib (10, 25 and 50nM), ponatinib (10, 50 and 100nM), asciminib) (ABL allosteric inhibitor) (5, 10 and 20nM) and ATO (0.5, 1.0 and 2.0 µM). We found that all the inhibitors significantly inhibited the proliferation of Ba/F3 cells expressing wild type BCR-ABL1 in a dose-dependent manner. In contrast, the growth of Ba/F3 cells expressing BCR-ABL1-T315I was inhibited by increasing concentration of ponatinib, asciminib and ATO. ATO potently inhibited the most challenging mutation (T315I-E255K) with a clinically relevant concentration (IC50 250nM). All approved ABL kinase inhibitors (AKIs) and allosteric inhibitors like asciminib could not inhibit the growth of Ba/F3 cells expressing BCR-ABL1 compound mutation. Conclusions: Our findings indicate that ATO significantly suppressed the proliferation of cells expressing non-mutated BCR-ABL1, single and compound mutated BCR-ABL1. These results support including ATO in treating patients with Ph + leukemias having BCR-ABL1 resistant single or compound mutati Disclosures Ottmann: Novartis: Honoraria; Amgen: Honoraria, Research Funding; Celgene/BMS: Honoraria, Research Funding; Fusion: Honoraria; Incyte: Honoraria, Research Funding.
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Meng, Kun, Jiang Li, Yanan Cao, Pengjun Shi, Bo Wu, Xiaoyu Han, Yingguo Bai, Ningfeng Wu, and Bin Yao. "Gene cloning and heterologous expression of a serine protease fromStreptomyces fradiaevar.k11." Canadian Journal of Microbiology 53, no. 2 (February 2007): 186–95. http://dx.doi.org/10.1139/w06-122.
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The gene sfp1, which encodes a predicted serine proteinase designated SFP1, was isolated by the screening of a gene library of the feather-degrading strain Streptomyces fradiae var.k11. The open reading frame of sfp1 encodes a protein of 454 amino acids with a calculated molecular mass of 46.19 kDa. Sequence analysis reveals that SFP1 possesses a typical pre-pro-mature organization that consists of a signal sequence, an N-terminal propeptide region, and a mature proteinase domain. The pre-enzyme of SFP1 was expressed in Escherichia coli and consequently purified. The 25.6 kDa fraction with protease activity separated by gel filtration chromatography indicated that the mature enzyme of SFP1 was formed by autolysis of the propeptide after its expression. The purified SFP1 is active under a broad range of pH and temperature. SFP1 has pH and temperature optima of pH 8.5 and 65 °C for its caseinolytic activity and pH 9 and 62 °C for its keratinolytic activity. SFP1 was sharply inhibited by the serine proteinase inhibitor phenylmethyl sulfonyl fluoride and exhibited a good stability to solvents, detergents, and salts. Comparison of the protease activity of SFP1 with other commercial proteases indicates that SFP1 has a considerable caseinolytic and keratinolytic activity as does proteinase K.
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Yokota, Asumi, Shinya Kimura, Satohiro Masuda, Eishi Ashihara, Junya Kuroda, Kiyoshi Sato, Yuri Kamitsuji, et al. "INNO-406, a novel BCR-ABL/Lyn dual tyrosine kinase inhibitor, suppresses the growth of Ph+ leukemia cells in the central nervous system, and cyclosporine A augments its in vivo activity." Blood 109, no. 1 (September 5, 2006): 306–14. http://dx.doi.org/10.1182/blood-2006-03-013250.
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Abstract Central nervous system (CNS) relapse accompanying the prolonged administration of imatinib mesylate has recently become apparent as an impediment to the therapy of Philadelphia chromosome–positive (Ph+) leukemia. CNS relapse may be explained by limited penetration of imatinib mesylate into the cerebrospinal fluid because of the presence of P-glycoprotein at the blood-brain barrier. To overcome imatinib mesylate–resistance mechanisms such as bcr-abl amplification, mutations within the ABL kinase domain, and activation of Lyn, we developed a dual BCR-ABL/Lyn inhibitor, INNO-406 (formerly NS-187), which is 25 to 55 times more potent than imatinib mesylate in vitro and at least 10 times more potent in vivo. The aim of this study was to investigate the efficacy of INNO-406 in treating CNS Ph+ leukemia. We found that INNO-406, like imatinib mesylate, is a substrate for P-glycoprotein. The concentrations of INNO-406 in the CNS were about 10% of those in the plasma. However, this residual concentration was enough to inhibit the growth of Ph+ leukemic cells which expressed not only wild-type but also mutated BCR-ABL in the murine CNS. Furthermore, cyclosporine A, a P-glycoprotein inhibitor, augmented the in vivo activity of INNO-406 against CNS Ph+ leukemia. These findings indicate that INNO-406 is a promising agent for the treatment of CNS Ph+ leukemia.
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Zuverink, Madison, Chen Chen, Amanda Przedpelski, Faith C. Blum, and Joseph T. Barbieri. "A Heterologous Reporter Defines the Role of the Tetanus Toxin Interchain Disulfide in Light-Chain Translocation." Infection and Immunity 83, no. 7 (April 20, 2015): 2714–24. http://dx.doi.org/10.1128/iai.00477-15.
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Botulinum neurotoxins (BoNTs) and tetanus toxin (TeNT) are the most potent toxins for humans and elicit unique pathologies due to their ability to traffic within motor neurons. BoNTs act locally within motor neurons to elicit flaccid paralysis, while retrograde TeNT traffics to inhibitory neurons within the central nervous system (CNS) to elicit spastic paralysis. BoNT and TeNT are dichain proteins linked by an interchain disulfide bond comprised of an N-terminal catalytic light chain (LC) and a C-terminal heavy chain (HC) that encodes an LC translocation domain (HCT) and a receptor-binding domain (HCR). LC translocation is the least understood property of toxin action, but it involves low pH, proteolysis, and an intact interchain disulfide bridge. Recently, Pirazzini et al. (FEBS Lett 587:150–155, 2013,http://dx.doi.org/10.1016/j.febslet.2012.11.007) observed that inhibitors of thioredoxin reductase (TrxR) blocked TeNT and BoNT action in cerebellar granular neurons. In the current study, an atoxic TeNT LC translocation reporter was engineered by fusing β-lactamase to the N terminus of TeNT [βlac-TeNT(RY)] to investigate LC translocation in primary cortical neurons and Neuro-2a cells. βlac-TeNT(RY) retained the interchain disulfide bond, showed ganglioside-dependent binding to neurons, required acidification to promote βlac translocation, and was sensitive to auranofin, an inhibitor of thioredoxin reductase. Mutation of βlac-TeNT(RY) at C439S and C467S eliminated the interchain disulfide bond and inhibited βlac translocation. These data support the requirement of an intact interchain disulfide for LC translocation and imply that disulfide reduction is a prerequisite for LC delivery into the host cytosol. The data also support a model that LC translocation proceeds from the C to the N terminus. βlac-TeNT(RY) is the first reporter system to measure translocation by an AB single-chain toxin in intact cells.
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Mian, Afsar, Anahita Rafiei, Anna Metodieva, Isabella Haberbosch, Alexey Zeifman, Ilya Titov, Victor Stroylov, et al. "PF-114, a Novel Selective Pan BCR/ABL Inhibitor Targets The T315I and Suppress Models Of Advanced Ph+ ALL." Blood 122, no. 21 (November 15, 2013): 3907. http://dx.doi.org/10.1182/blood.v122.21.3907.3907.
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Abstract Background Targeting BCR/ABL by ABL-directed kinase inhibitors (AKIs) induces long lasting remissions in patients with chronic myeloid leukemia (CML), and short remissions in Ph+ acute lymphatic leukemia (ALL). Notably in advanced Ph+ leukemia resistance attributable to either kinase domain mutations in BCR/ABL or non mutational mechanisms remains the major clinical challenge. With the only exception of Ponatinib, a multitargeted kinase inhibitor, all actually approved AKIs are unable to inhibit the „gatekeeper“ mutation T315I. Ponatinib is unable to overcome non mutational resistance in advanced leukemia and has an unfavorable spectrum of undesired side effects, most likely due to the broad spektrum of kinase inhibition. Thus there is the urgent need for further and more selective therapeutical options in treating therapy-resistant advanced Ph+ leukemia. PF114 is an ATP competitor, which was developed focusing on: i.) targeting all known resistance mutations in BCR/ABL but mainly the T315I; ii.) a higher selectivity as compared to Ponatinib in order to reduce undesired side effects; iii.) the ability to overcome also non mutational resistance in advanced Ph+ leukemia; iv.) an activity not only in chronic phase CML but also advanced Ph+ leukemia, Ph+ ALL or blast crisis CML (BC-CML). Methods An toxicity profile of PF-114 as well as its kinase selectivity was investigated. The preclinical evaluation of PF-114 was performed in direct comparison to Ponatinib on golden standard preclinical models of CML and advanced Ph+ leukemia and primary patient-derived long term cultures (PD-LTC) of Ph+ ALL patients. The effects on mutational resistance was investigated i.) on the factor dependence of Ba/F3 cells expressing BCR/ABL or its clinically most relevant resistance mutants (Y253F, E255K, T315I, F317L); ii.) on a PD-LTC of a Ph+ ALL patient harboring the T315I. As models for non mutational resistance we used PD-LTCs from Ph+ ALL patients with different levels of non mutational drug resistance and the SupB15RT, a Ph+ ALL cell line rendered resistant by the exposure to increasing doses of Imatinib and cross-resistant against all approved AKIs. The effects of PF114 in vivo were investigated on the transduction/transplantation model of BCR/ABL- and BCR/ABL-T315I-induced CML-like disease, secondary BCR/ABL-induced murine ALLs as well as on xenografts of PD-LTCs in NSG mice and K562 cells in nude mice. Results PF-114 is an orally available AKI, which is more selective than Dasatinib or Ponatinib (number of kinases inhibited at 100 nM of a drug: Nilotinib - 19; PF-114 - 27; Dasatinib - 48; Ponatinib - 80). It was classified as a class 4 - low-toxic - substance (Hodge/Sterner classification). It efficiently inhibited all tested BCR/ABL mutants in cellular and biochemical assays at dosages of 10-100nM and like Ponatinib it suppressed the up-coming of new resistance mutations in a mutation assay in Ba/F3 cells. It also suppressed growth of Ph+ PD-LTC with non mutational resistance as well as the BCR/ABL-T315I-positve PD-LTC in this dosage range. In all models the effect was independent of the presence of either the p210 or the p185 form of BCR/ABL. No effect of PF114 was seen in PD-LTCs of Ph- ALL. Noteworthy PF-114 (50 mg/Kg) prolonged significantly the survival of mice with both BCR/ABL- and BCR/ABL-T315I-driven CML-like disease as compared to Ponatinib (25 mg/Kg). In all in vivo models of advanced leukemia, PF-114 (50 mg/Kg) significantly inhibited leukemia to a similar extent as Ponatinib (25 mg/Kg). Like Ponatinib, PF-114 was unable to overcome the non mutational resistance in SupB15RT. Conclusions Our work supports clinical evaluation of PF114 as a pan BCR/ABL inhibitor for treatment not only for chronic phase CML, but also for advanced and resistant Ph+ leukemia such as Ph+ ALL or BC-CML. Disclosures: Mian: Fusion Pharma: Research Funding. Zeifman:Fusion Pharma: Employment. Titov:Fusion Pharma: Employment. Stroylov:Fusion Pharma: Employment. Stroganov:Fusion Pharma: Employment. Novikov:Fusion Pharma: Employment. Chilov:Fusion Pharma: Employment. Ruthardt:Fusion Pharma: Research Funding.
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Deraison, Celine, Chrystelle Bonnart, Frederic Lopez, Celine Besson, Ross Robinson, Arumugam Jayakumar, Fredrik Wagberg, et al. "LEKTI Fragments Specifically Inhibit KLK5, KLK7, and KLK14 and Control Desquamation through a pH-dependent Interaction." Molecular Biology of the Cell 18, no. 9 (September 2007): 3607–19. http://dx.doi.org/10.1091/mbc.e07-02-0124.
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LEKTI is a 15-domain serine proteinase inhibitor whose defective expression underlies the severe autosomal recessive ichthyosiform skin disease, Netherton syndrome. Here, we show that LEKTI is produced as a precursor rapidly cleaved by furin, generating a variety of single or multidomain LEKTI fragments secreted in cultured keratinocytes and in the epidermis. The identity of these biological fragments (D1, D5, D6, D8–D11, and D9–D15) was inferred from biochemical analysis, using a panel of LEKTI antibodies. The functional inhibitory capacity of each fragment was tested on a panel of serine proteases. All LEKTI fragments, except D1, showed specific and differential inhibition of human kallikreins 5, 7, and 14. The strongest inhibition was observed with D8–D11, toward KLK5. Kinetics analysis revealed that this interaction is rapid and irreversible, reflecting an extremely tight binding complex. We demonstrated that pH variations govern this interaction, leading to the release of active KLK5 from the complex at acidic pH. These results identify KLK5, a key actor of the desquamation process, as the major target of LEKTI. They disclose a new mechanism of skin homeostasis by which the epidermal pH gradient allows precisely regulated KLK5 activity and corneodesmosomal cleavage in the most superficial layers of the stratum corneum.
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von Bubnoff, Nikolas, Darren R. Veach, Heiko van der Kuip, Walter E. Aulitzky, Jana Saenger, Petra Seipel, William G. Bornmann, Christian Peschel, Bayard Clarkson, and Justus Duyster. "A Cell-Based Screening Method for Resistance of Bcr-Abl Positive Leukemia Identifies the Mutation Pattern for an Alternative Abl Kinase Inhibitor." Blood 104, no. 11 (November 16, 2004): 558. http://dx.doi.org/10.1182/blood.v104.11.558.558.
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Abstract The increasing impact of targeted cancer treatment demands strategies to identify and evaluate resistance mechanisms toward kinase inhibitors prior to their therapeutic application. Point mutations within the Bcr-Abl kinase domain constitute the major mechanism of resistance toward imatinib mesylate in Philadelphia-positive (Ph+) leukemia. Using Bcr-Abl-transformed Ba/F3 cells, we established a cell-based screening strategy for the prediction of specific kinase mutations that cause resistance toward kinase inhibitors. With imatinib at clinically relevant concentrations, we generated 368 resistant Ba/F3 sublines that were derived from resistant colonies. Thirty-two different single point mutations within the kinase domain of Bcr-Abl were identified in twenty-five per cent (liquid culture conditions) and seventy-two per cent (solid culture conditions) of these lines at known and novel positions. Using imatinib, the pattern and relative frequency of mutations reflected matters observed in patients with imatinib resistance. We then applied this screen to the pyrido-pyrimidine PD166326 (PD16), an investigational Abl kinase inihibitor. Compared to imatinib, we observed a five to seven times lower frequency of resistant colonies with equipotent concentrations of PD16. In addition, PD16 produced a distinct pattern of Bcr-Abl mutations. P-loop, A-loop and the known imatinib contact site T315 were affected with both inhibitors, whereas C-helix and SH2 contact sites were affected in imatinib resistant colonies exclusively. In contrast to imatinib, where kinase domain mutations were still widely distributed over the kinase domain even at at 4μM, mutations observed with PD16 at a concentration of 100nM narrowed to the exchange at position T315 to iseulicine. We did not detect mutations outside the kinase domain. Some resistant sublines displayed increased Bcr-Abl activity. Mutations that were derived from the screen were cloned and examined for the extent of cross-resistance to both inhibitors. The majority of mutations were effectively suppressed by PD16 at 50–500nM. In contrast, only few mutations were inhibited by imatinib at 5–10μM. However, exchanges at position F317 mediated resistance toward PD16, but were inhibited by standard concentrations of imatinib. Since this cell-based system produced results that are clinically significant, it may be used to predict resistance mutations in Bcr-Abl and other oncogenic kinases like cKit, EGFR, FIP1L1-PDGFRalpha or FLT3 towards clinically applicated and investigational drugs. Thus, this robust and simple screening strategy provides a rational basis for combinatorial and sequential treatment strategies.
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Hoffmann, Markus, Svenja Victoria Kaufmann, Carina Fischer, Wiebke Maurer, Anna-Sophie Moldenhauer, and Stefan Pöhlmann. "Analysis of Resistance of Ebola Virus Glycoprotein-Driven Entry Against MDL28170, An Inhibitor of Cysteine Cathepsins." Pathogens 8, no. 4 (October 15, 2019): 192. http://dx.doi.org/10.3390/pathogens8040192.
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Ebola virus (EBOV) infection can cause severe and frequently fatal disease in human patients. The EBOV glycoprotein (GP) mediates viral entry into host cells. For this, GP depends on priming by the pH-dependent endolysosomal cysteine proteases cathepsin B (CatB) and, to a lesser degree, cathepsin L (CatL), at least in most cell culture systems. However, there is limited information on whether and how EBOV-GP can acquire resistance to CatB/L inhibitors. Here, we addressed this question using replication-competent vesicular stomatitis virus bearing EBOV-GP. Five passages of this virus in the presence of the CatB/CatL inhibitor MDL28170 were sufficient to select resistant viral variants and sequencing revealed that all GP sequences contained a V37A mutation, which, in the context of native GP, is located in the base of the GP surface unit. In addition, some GP sequences harbored mutation S195R in the receptor-binding domain. Finally, mutational analysis demonstrated that V37A but not S195R conferred resistance against MDL28170 and other CatB/CatL inhibitors. Collectively, a single amino acid substitution in GP is sufficient to confer resistance against CatB/CatL inhibitors, suggesting that usage of CatB/CatL inhibitors for antiviral therapy may rapidly select for resistant viral variants.
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Connor, Lisa M. O’, Stephen Langabeer, Shaun R. McCann, and Eibhlin Conneally. "Mutation Mediated Resistance to the Tyrosine Kinase Inhibitors Imatinib, Dasatinib & Nilotinib in Philadelphia Positive Leukaemia." Blood 112, no. 11 (November 16, 2008): 4245. http://dx.doi.org/10.1182/blood.v112.11.4245.4245.
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Abstract The Philadelphia chromosome is formed as a result of a reciprocal translocation between chromosomes 9 and 22 and results in the formation of the hybrid oncoprotein BCR-ABL. It is observed in over 95% of Chronic Myeloid Leukaemia (CML) and approximately 30% of adult Acute Lymphoblastic Leukaemia (ALL) cases. Imatinib Mesylate (IM), a tyrosine kinase inhibitor that specifically binds BCR-ABL in its inactive conformation has revolutionized therapy for CML and Ph+ ALL. However, resistance develops in a significant proportion of patients and is predominantly mediated by single base-pair substitutions within the BCR-ABL kinase domain leading to changes in the amino acid composition that inhibit IM binding whilst retaining BCR-ABL phosphorylation capacity. Second generation tyrosine kinase inhibitors such as Dasatinib and Nilotinib retain activity in IM-resistant patients due to less stringent binding requirements and represent viable alternatives for IM-resistant patients with a suitable molecular profile. In this study, we undertook to examine the molecular mechanisms underlying IM resistance. A cohort of 40 patients with either primary or acquired resistance or intolerance to IM was identified by persistent high or increasing levels of BCR-ABL transcripts determined by real-time quantitative PCR. An allele-specific PCR screen was used to sensitively detect the clinically significant T315I mutation, which renders patients insensitive to currently available tyrosine kinase inhibitors: five (12.5%) IM resistant/intolerant patients were T315I positive. To further elucidate the molecular mechanisms of mutation induced resistance, the BCR-ABL kinase domain was screened for the presence of a mutation using a sensitive denaturing high performance liquid chromatography (dHPLC) approach. Samples showing evidence of mutation were examined by direct sequencing to identify the mutation(s) present. Kinase domain mutations have been identified in 20 of the 40 (50%) patients examined to date and these include p-loop mutations (M244V, G250E, Q252H), IM-binding domain mutations (T315I), catalytic domain mutations (M351T), an activation-loop mutation (L387M). Three previously unreported mutations were identified in patients with indications of IM resistance (T267A, E275Q) and Nilotinib resistance (L273M). The L273 residue lies adjacent to a region of the BCR-ABL kinase domain bound by Nilotinib. Three patients were found to harbour mutations at two distinct kinase domain residues while one patient harboured mutations at three distinct residues, supporting the theory that patients who develop mutation-mediated resistance to one kinase inhibitor may become resistant to subsequent inhibitors by a similar mechanism. The identification of clinically significant mutations facilitates selection of alternative approaches to therapy such as dose escalation of IM, second generation tyrosine kinase inhibitors or allogeneic stem cell transplant, if eligible, at an early stage in a patient’s disease, tailoring patient specific approaches to therapy.
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Quentmeier, Hilmar, Sonja Eberth, Julia Romani, Margarete Zaborski, and Hans G. Drexler. "Induction of Apoptosis In Imatinib-Resistant BCR-ABL1-Positive Leukemia Cell Lines by Bortezomib." Blood 116, no. 21 (November 19, 2010): 4469. http://dx.doi.org/10.1182/blood.v116.21.4469.4469.
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Abstract Abstract 4469 The BCR-ABL1 translocation occurs in chronic myeloid leukemia (CML) and in 25% of cases with acute lymphoblastic leukemia (ALL). We screened a panel of BCR-ABL1 positive cell lines to find models for imatinib-resistance studies. Five of 19 BCR-ABL1 positive cell lines were resistant to imatinib-induced apoptosis (KCL-22, MHH-TALL1, NALM-1, SD-1, SUP-B15). None of the five resistant cell lines carried mutations in the kinase domain of BCR-ABL1 and – consequently – all also showed resistance to the second generation kinase inhibitors, nilotinib or dasatinib. All Philadelphia chromosome (Ph)-positive cell lines demonstrated constitutive phosphorylation of STAT5 and S6. Imatinib induced dephosphorylation of both BCR-ABL1 downstream effectors in responsive cell lines, but - remarkably – induced dephosphorylation of STAT5 in resistant cell lines as well. By administering well-described signalling pathway inhibitors we were able to show that activation of mTOR complex 1 was responsible for the constitutive S6 phosphorylation of imatinib-resistant cells. Neither BCR-ABL1 nor Src kinases or Ras/Rac-GTPases underlie tyrosine kinase inhibitor resistance in these cell lines. In conclusion, none of the five TKI-resistant cell lines showed aberrant activation of previously-described oncogenic pathways which would explain their resistance. These findings raise the question whether these cell lines might help to find a novel – alternative – explanation for TKI resistance. Interestingly, the proteasome inhibitor bortezomib induced apoptosis in TKI-resistant and –sensitive Ph+ cell lines. Bortezomib is being used for the treatment of multiple myeloma. Our findings support the notion that bortezomib might also be useful for the treatment of imatinib-resistant CML. Disclosures: No relevant conflicts of interest to declare.
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Roberts, Kathryn G., Ryan D. Morin, Jinghui Zhang, Martin Hirst, Richard C. Harvey, Corynn Kasap, Xiang Chen, et al. "Novel Chromosomal Rearrangements and Sequence Mutations in High-Risk Ph-Like Acute Lymphoblastic Leukemia." Blood 118, no. 21 (November 18, 2011): 67. http://dx.doi.org/10.1182/blood.v118.21.67.67.
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Abstract Abstract 67 Acute lymphoblastic leukemia (ALL) is the most common pediatric malignancy, and relapsed B-lineage ALL remains a leading cause of cancer death in young people. Recent genomic analyses by our group and others identified a unique subtype of BCR-ABL-negative, high-risk B-ALL, with deletion or mutation of IKZF1 and a gene expression profile similar to BCR-ABL1-positive ALL (Ph-like ALL). Up to 50% of Ph-like patients harbor rearrangements of the cytokine receptor gene, CRLF2, with concomitant JAK mutations detected in ∼30%. However, the nature of genetic alterations activating kinase signaling in the remaining cases is unknown. To identify novel genetic alterations in Ph-like ALL, we performed transcriptome sequencing (RNA-seq) on 11 cases of Ph-like B-ALL (10 from the P9906 Children's Oncology Group trial and 1 from the St Jude Total XV study), and whole genome sequencing (WGS) on two of these. Using multiple complementary analysis pipelines including deFuse, Mosaik, CREST and CONSERTING, we identified novel rearrangements, structural variations and sequence mutations dysregulating cytokine receptor and kinase signaling in 10 cases. Putative rearrangements and sequence mutations were validated using RT-PCR, genomic PCR and Sanger sequencing. The spectrum of alterations included 3 cases with known IGH@CRLF2 rearrangement, 2 cases with the NUP214-ABL1 rearrangement, 1 case each with the in-frame fusions EBF1-PDGFRB, BCR-JAK2 or STRN3-JAK2, and 1 case with a cryptic IGH@-EPOR rearrangement. Detailed analysis of RNA-seq data revealed a 7.5 kb insertion of EPOR downstream of the enhancer domain in the IGH@ locus, which was not detected by fluorescence in situ hybridization. WGS identified an in-frame activating insertion in the transmembrane domain of IL7R (L242>FPGVC) in 1 index case, and recurrence screening identified similar IL7R sequence mutations in 8 cases from the P9906 cohort (N=188). This patient also harbored a focal homozygous deletion removing the first two exons of SH2B3 that was not evident by SNP array analysis. SH2B3 encodes LNK, a negative regulator of JAK2 signaling. Notably, all patients harbor genetic lesions affecting B-lymphoid development (e.g IKZF1), suggesting these events cooperate to drive B-lineage ALL. To determine the frequency of each fusion, candidate RT-PCR was performed on 231 cases from the COG AALL0232 trial of high-risk B-ALL, 40 (17%) of which were identified as Ph-like using Predictor Analysis of Microarrays (PAM). The EBF1-PDGFRB fusion was detected in 3 additional patients, each containing an intact PDGFRB kinase domain. No additional cases of NUP214-ABL1, BCR-JAK2, or STRN3-JAK2 were identified. Phosphoflow analysis on 3 primary ALL samples demonstrated increased CKRL phosphorylation in the NUP214-ABL1 case and tyrosine phosphorylation in the cases with BCR-JAK2 and STRN3-JAK2 fusions. Importantly, this activation was reduced with the tyrosine kinase inhibitors (TKI) imatinib, dasatinib and the T315I inhibitor XL228 in cells harboring the ABL1 fusion, and the JAK2 inhibitor, XL019, in the JAK2-rearranged samples. Furthermore, the novel EBF1-PDGFRB fusion transformed Ba/F3 cells to growth factor independence, induced constitutive activation of pSTAT5, pAkt, pERK1/2, and responded with low IC50 values to imatinib, dasatinib and the specific PDGFRB/FGFR inhibitor, dovitinib. Using complementary genomic approaches we show that rearrangements, sequence mutations and DNA copy number alterations dysregulating cytokine receptor and kinase signaling are a hallmark of Ph-like ALL. These data support the screening of patients at diagnosis to identify those with Ph-like ALL, characterize the genomic lesions driving this phenotype, and to determine those that may benefit from TKI treatment. Disclosures: Hunger: Bristol-Myers Squibb: Author's children own stock in BMS, Membership on an entity's Board of Directors or advisory committees.
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Vg, Meenu Krishnan, and Murugan K. "SOLANUM PROTEASE INHIBITORS AND THEIR THERAPEUTIC POTENTIALITIES: A REVIEW." International Journal of Pharmacy and Pharmaceutical Sciences 8, no. 12 (December 1, 2016): 14. http://dx.doi.org/10.22159/ijpps.2016v8i12.14836.
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<p>Protease inhibitors (PIs) are diverse group of proteins with low molecular weight that are ubiquitous in all life forms. PIs are reducers of the physiological activity of proteases and fascinated the attention of biotechnological researchers. In the evolutionary course, plants have developed diverse adaptive mechanisms of defence against various unfavorable conditions including that of predators and pathogens. Phylogenetic relationships among diverse PI families like serpin, Bowman–Birk, cereal trypsin/α-amylase inhibitor, proteinase inhibitor I, proteinase inhibitor II and cystatin have been evaluated. PIs evolution seems to occur through multiple interacting mechanisms and not commonly seen with other co-evolving molecules. Interaction of PIs produced by host organisms and the invasive/dietary protease of pathogens or parasites or predators, leads to a phylogenetic ‘arms race’ of rapid structural modulation in both interacting proteins. Further, the high rate of retention of gene duplication and inhibitory domain multiplication results the PI as potential model system to trace the basic evolutionary process of functional diversification. The mode of action of PI is either <em>via</em> inactivating the hydrolytic enzymes or depolarization of cell membrane of the pathogens thereby inhibiting its growth and invasion. Generally, PIs possess significant number of disulfide bonds due to cysteine residues that provide them resistance to heat, extremes of pH, and proteolysis. However, PIs have been extracted and purified only from few monocots and dicots plants. Currently, PI genes were used for developing insect-resistant transgenic plants for crop improvements. Classification of PIs over the last several years has been based on structural–functional relationships. This review bridges the gap between the folkloric uses of <em>Solanum </em>PIs, their diversity and biological potentialities.</p>
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Hantschel, Oliver D., Florian Grebien, Ines Kaupe, Boris Kovacic, John Wojcik, Gerald D. Gish, Shohei Koide, Hartmut Beug, Tony Pawson, and Giulio Superti-Furga. "The Bcr-Abl SH2-Kinase Domain Interface Is Critical for Leukemogenesis and An Additional Therapeutic Target in CML." Blood 114, no. 22 (November 20, 2009): 37. http://dx.doi.org/10.1182/blood.v114.22.37.37.
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Abstract Abstract 37 We previously showed that the Abl SH2 domain is an allosteric activator of c-Abl tyrosine kinase activity and substrate phosphorylation (Filippakopoulos et al. (2008) Cell 134(5), 793-803). This effect is exerted directly by docking of the SH2 domain onto the N-lobe of the kinase domain in the active conformation of c-Abl. We also showed that the same structural mechanism is a critical factor for full activation of the oncogenic fusion kinase Bcr-Abl. Disruption of binding of the SH2 domain to the kinase domain in Bcr-Abl by the Ile164Glu mutation in the SH2 domain, led to a strong reduction in in vitro tyrosine kinase activity and Bcr-Abl autophosphorylation. Unexpectedly, we observed a differential attenuation of downstream signaling pathways upon disruption of the SH2-kinase domain interface, indicating different activation thresholds of Bcr-Abl downstream signaling pathways. Here, we show that disrupting the SH2-kinase domain interface abrogates the transforming capacity of Bcr-Abl. Cells expressing the Bcr-Abl Ile164Glu mutant were unable to generate cytokine-independent colonies in vitro. Furthermore, mice transplanted with Bcr-Abl Ile164Glu expressing bone marrow cells did not develop the characteristic MPD-like disease that is caused by wild-type Bcr-Abl. Mice that received Bcr-Abl Ile164Glu cells showed normal survival, blood counts and histology after more than 100 days post-transplant, despite the presence of Bcr-Abl Ile164Glu-expressing cells in all blood lineages. This shows that the formation of the SH2-kinase domain interface is strictly necessary for Bcr-Abl to cause CML. Together with our data that show sensitization to imatinib inhibition of Bcr-Abl Ile164Glu as compared to Bcr-Abl wild-type, this argues for the SH2-kinase domain interface as an additional drug target on Bcr-Abl that may synergize with tyrosine kinase inhibitors and may be useful to inhibit tyrosine kinase inhibitor resistant Bcr-Abl clones. To address possibilities to interfere with the SH2-kinase domain interface, we are using an engineered binding protein that binds to the Abl SH2 domain with high-affinity and specificity and supposedly disrupts the interface with the kinase domain, resulting in a decrease in Bcr-Abl kinase activity. In conclusion, we provide strong evidence that the structural positioning of the SH2 domain is a crucial factor for constitutive activity, signal transduction and leukemogenicity of Bcr-Abl. Besides oligomerization via the N-terminal coiled-coiled domain and loss of the auto-inhibitory N-terminal myristoyl group, the proper positioning of the SH2 domain appears to be another critical factor that is required for constitutive activation of Bcr-Abl. Inhibitors of the SH2-kinase domain interface of Bcr-Abl may comprise alternative or additional points of pharmacological intervention for the treatment of imatinib-sensitive or -resistant CML or Ph+ acute lymphocytic leukemia. Disclosures: No relevant conflicts of interest to declare.
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Uko, Nne E., Osman F. Güner, Diane F. Matesic, and J. Phillip Bowen. "Akt Pathway Inhibitors." Current Topics in Medicinal Chemistry 20, no. 10 (May 19, 2020): 883–900. http://dx.doi.org/10.2174/1568026620666200224101808.
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Cancer is a devastating disease that has plagued humans from ancient times to this day. After decades of slow research progress, promising drug development, and the identification of new targets, the war on cancer was launched, in 1972. The P13K/Akt pathway is a growth-regulating cellular signaling pathway, which in many human cancers is over-activated. Studies have demonstrated that a decrease in Akt activity by Akt inhibitors is associated with a reduction in tumor cell proliferation. There have been several promising drug candidates that have been studied, including but not limited to ipatasertib (RG7440), 1; afuresertib (GSK2110183), 2; uprosertib (GSK2141795), 3; capivasertib (AZD5363), 4; which reportedly bind to the ATP active site and inhibit Akt activity, thus exerting cytotoxic and antiproliferative activities against human cancer cells. For most of the compounds discussed in this review, data from preclinical studies in various cancers suggest a mechanistic basis involving hyperactivated Akt signaling. Allosteric inhibitors are also known to alter the activity of kinases. Perifosine (KRX- 0401), 5, an alkylphospholipid, is known as the first allosteric Akt inhibitor to enter clinical development and is mechanistically characterized as a PH-domain dependent inhibitor, non-competitive with ATP. This results in a reduction in Akt enzymatic and cellular activities. Other small molecule (MK- 2206, 6, PHT-427, Akti-1/2) inhibitors with a similar mechanism of action, alter Akt activity through the suppression of cell growth mediated by the inhibition of Akt membrane localization and subsequent activation. The natural product solenopsin has been identified as an inhibitor of Akt. A few promising solenopsin derivatives have emerged through pharmacophore modeling, energy-based calculations, and property predictions.
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Vilella, S., V. Zonno, S. Marsigliante, L. Ingrosso, A. Muscella, M. M. Ho, G. P. Vinson, and C. Storelli. "Angiotensin II stimulation of the basolateral located Na+/H+ antiporter in eel (Anguilla anguilla) enterocytes." Journal of Molecular Endocrinology 16, no. 1 (February 1996): 57–62. http://dx.doi.org/10.1677/jme.0.0160057.
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ABSTRACT The pH-sensitive fluorescent dye, 2′,7′-bis-carboxy-ethyl-5,6-carboxyfluorescein acetoxymethyl ester, was used to examine the effects of fish or human angiotensin II (Ang II) on the activity of the basolateral located Na+/H+ antiporter in eel intestinal cell suspensions. Exposure of eel enterocytes to either hormone led to an increased activity of the antiporter. This time- and dose-dependent stimulatory effect was inhibited by the specific antiporter inhibitor dimethylamiloride (DMA). Preincubation with a monoclonal antibody (6313/G2), directed against the N-terminal extracellular domain of the mammalian AT1 Ang II receptor, prevented the stimulatory effect of the hormone and inhibited the binding of [3,5-3H]Tyr4-Ile5-Ang II to intestinal cell suspensions, suggesting specific binding of the antibody to the eel Ang II receptor. The results indicate that both fish and human Ang II stimulate the DMA-sensitive Na+/H+ antiporter present in eel intestinal cells by means of a mammalian AT1-like receptor.