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ABSTRACT: The upstream protein kinases responsible for thousands of phosphorylation events in the phosphoproteome remain to be discovered. We developed a three-component chemical reaction which converts the transient noncovalent substrate-kinase complex into a covalently cross-linked product by utilizing a dialdehyde-based cross-linker, 1. Unfortunately, the reaction of 1 with a lysine in the kinase active site and an engineered cysteine on the substrate to form an isoindole cross-linked product could not be performed in the presence of competing cellular proteins due to nonspecific side reactions. In order to more selectively target the cross-linker to protein kinases in cell lysates, we replaced the weak, kinase-binding adenosine moiety of 1 with a potent protein kinase inhibitor scaffold. In addition, we replaced the o-phthaldialdehyde moiety in 1 with a less-reactive thiophene-2,3-dicarboxaldehyde moiety. The combination of these two structural modifications provides for cross-linking of a cysteine-containing substrate to its corresponding kinase in the presence of competing cellular proteins.
Journal of the American Chemical Society 12/2008; 130(51):17568-74. · 9.91 Impact Factor
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Matthew A Young,
Neil P Shah,
Luke H Chao,
Markus Seeliger,
Zdravko V Milanov, William H Biggs,
Daniel K Treiber,
Hitesh K Patel,
Patrick P Zarrinkar,
David J Lockhart,
Charles L Sawyers,
John Kuriyan
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ABSTRACT: We present a high-resolution (2.0 A) crystal structure of the catalytic domain of a mutant form of the Abl tyrosine kinase (H396P; Abl-1a numbering) that is resistant to the Abl inhibitor imatinib. The structure is determined in complex with the small-molecule inhibitor VX-680 (Vertex Pharmaceuticals, Cambridge, MA), which blocks the activity of various imatinib-resistant mutant forms of Abl, including one (T315I) that is resistant to both imatinib and BMS-354825 (dasatinib), a dual Src/Abl inhibitor that seems to be clinically effective against all other imatinib-resistant forms of BCR-Abl. VX-680 is shown to have significant inhibitory activity against BCR-Abl bearing the T315I mutation in patient-derived samples. The Abl kinase domain bound to VX-680 is not phosphorylated on the activation loop in the crystal structure but is nevertheless in an active conformation, previously unobserved for Abl and inconsistent with the binding of imatinib. The adoption of an active conformation is most likely the result of synergy between the His(396)Pro mutation, which destabilizes the inactive conformation required for imatinib binding, and the binding of VX-680, which favors the active conformation through hydrogen bonding and steric effects. VX-680 is bound to Abl in a mode that accommodates the substitution of isoleucine for threonine at residue 315 (the "gatekeeper" position). The avoidance of the innermost cavity of the Abl kinase domain by VX-680 and the specific recognition of the active conformation explain the effectiveness of this compound against mutant forms of BCR-Abl, including those with mutations at the gatekeeper position.
Cancer Research 02/2006; 66(2):1007-14. · 7.86 Impact Factor
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Todd A Carter,
Lisa M Wodicka,
Neil P Shah,
Anne Marie Velasco,
Miles A Fabian,
Daniel K Treiber,
Zdravko V Milanov,
Corey E Atteridge, William H Biggs,
Philip T Edeen, [......],
Robert M Grotzfeld,
Sanna Herrgard,
Darren E Insko,
Shamal A Mehta,
Hitesh K Patel,
William Pao,
Charles L Sawyers,
Harold Varmus,
Patrick P Zarrinkar,
David J Lockhart
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ABSTRACT: To realize the full potential of targeted protein kinase inhibitors for the treatment of cancer, it is important to address the emergence of drug resistance in treated patients. Mutant forms of BCR-ABL, KIT, and the EGF receptor (EGFR) have been found that confer resistance to the drugs imatinib, gefitinib, and erlotinib. The mutations weaken or prevent drug binding, and interestingly, one of the most common sites of mutation in all three kinases is a highly conserved "gatekeeper" threonine residue near the kinase active site. We have identified existing clinical compounds that bind and inhibit drug-resistant mutant variants of ABL, KIT, and EGFR. We found that the Aurora kinase inhibitor VX-680 and the p38 inhibitor BIRB-796 inhibit the imatinib- and BMS-354825-resistant ABL(T315I) kinase. The KIT/FLT3 inhibitor SU-11248 potently inhibits the imatinib-resistant KIT(V559D/T670I) kinase, consistent with the clinical efficacy of SU-11248 against imatinib-resistant gastrointestinal tumors, and the EGFR inhibitors EKB-569 and CI-1033, but not GW-572016 and ZD-6474, potently inhibit the gefitinib- and erlotinib-resistant EGFR(L858R/T790M) kinase. EKB-569 and CI-1033 are already in clinical trials, and our results suggest that they should be considered for testing in the treatment of gefitinib/erlotinib-resistant non-small cell lung cancer. The results highlight the strategy of screening existing clinical compounds against newly identified drug-resistant mutant variants to find compounds that may serve as starting points for the development of next-generation drugs, or that could be used directly to treat patients that have acquired resistance to first-generation targeted therapy.
Proceedings of the National Academy of Sciences 09/2005; 102(31):11011-6. · 9.68 Impact Factor
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Miles A Fabian, William H Biggs,
Daniel K Treiber,
Corey E Atteridge,
Mihai D Azimioara,
Michael G Benedetti,
Todd A Carter,
Pietro Ciceri,
Philip T Edeen,
Mark Floyd, [......],
Michael A Insko,
Andiliy G Lai,
Jean-Michel Lélias,
Shamal A Mehta,
Zdravko V Milanov,
Anne Marie Velasco,
Lisa M Wodicka,
Hitesh K Patel,
Patrick P Zarrinkar,
David J Lockhart
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ABSTRACT: Kinase inhibitors show great promise as a new class of therapeutics. Here we describe an efficient way to determine kinase inhibitor specificity by measuring binding of small molecules to the ATP site of kinases. We have profiled 20 kinase inhibitors, including 16 that are approved drugs or in clinical development, against a panel of 119 protein kinases. We find that specificity varies widely and is not strongly correlated with chemical structure or the identity of the intended target. Many novel interactions were identified, including tight binding of the p38 inhibitor BIRB-796 to an imatinib-resistant variant of the ABL kinase, and binding of imatinib to the SRC-family kinase LCK. We also show that mutations in the epidermal growth factor receptor (EGFR) found in gefitinib-responsive patients do not affect the binding affinity of gefitinib or erlotinib. Our results represent a systematic small molecule-protein interaction map for clinical compounds across a large number of related proteins.
Nature Biotechnology 04/2005; 23(3):329-36. · 23.27 Impact Factor
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ABSTRACT: Genetic analysis in Caenorhabditis elegans has uncovered essential roles for DAF-16 in longevity, metabolism, and reproduction. The mammalian orthologs of DAF-16, the closely-related FOXO subclass of forkhead transcription factors (FKHR/FOXO1, FKHRL1/FOXO3a, and AFX/FOXO4), also have important roles in cell cycle arrest, apoptosis and stress responses in vitro, but their in vivo physiological roles are largely unknown. To elucidate their role in normal development and physiology, we disrupted each of the Foxo genes in mice. Foxo1-null embryos died on embryonic day 10.5 as a consequence of incomplete vascular development. Foxo1-null embryonic and yolk sac vessels were not well developed at embryonic day 9.5, and Foxo1 expression was found in a variety of embryonic vessels, suggesting a crucial role of this transcription factor in vascular formation. On the other hand, both Foxo3a- and Foxo4-null mice were viable and grossly indistinguishable from their littermate controls, indicating dispensability of these two members of the Foxo transcription factor family for normal vascular development. Foxo3a-null females showed age-dependent infertility and had abnormal ovarian follicular development. In contrast, histological analyses of Foxo4-null mice did not identify any consistent abnormalities. These results demonstrate that the physiological roles of Foxo genes are functionally diverse in mammals.
Proceedings of the National Academy of Sciences 04/2004; 101(9):2975-80. · 9.68 Impact Factor
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ABSTRACT: Human SSX was first identified as the gene involved in the t(X;18) translocation in synovial sarcoma. SSX is a multigene family, with 9 complete genes on chromosome Xp11. Normally expressed almost exclusively in testis, SSX mRNA is expressed in various human tumors, defining SSX as a cancer/testis antigen. We have now cloned the mouse ortholog of SSX. Mouse SSX genes can be divided into Ssxa and Ssxb subfamilies based on sequence homology. Ssxa has only one member, whereas 12 Ssxb genes, Ssxb1 to Ssxb12, were identified by cDNA cloning from mouse testis and mouse tumors. Both Ssxa and Ssxb are located on chromosome X and show tissue-restricted mRNA expression to testis among normal tissues. All putative human and mouse SSX proteins share conserved KRAB and SSX-RD domains. Mouse tumors were found to express some, but not all, Ssxb genes, similar to the SSX activation in human tumors.
Genomics 01/2004; 82(6):628-36. · 3.02 Impact Factor
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ABSTRACT: An outstanding question in adipocyte biology is how hormonal cues are relayed to the nucleus to activate the transcriptional program that promotes adipogenesis. The forkhead transcription factor Foxo1 is regulated by insulin via Akt-dependent phosphorylation and nuclear exclusion. We show that Foxo1 is induced in the early stages of adipocyte differentiation but that its activation is delayed until the end of the clonal expansion phase. Constitutively active Foxo1 prevents the differentiation of preadipocytes, while dominant-negative Foxo1 restores adipocyte differentiation of fibroblasts from insulin receptor-deficient mice. Further, Foxo1 haploinsufficiency protects from diet-induced diabetes in mice. We propose that Foxo1 plays an important role in the integration of hormone-activated signaling pathways with the complex transcriptional cascade that promotes adipocyte differentiation.
Developmental Cell 02/2003; 4(1):119-29. · 14.03 Impact Factor
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ABSTRACT: Diabetes is caused by an absolute (type 1) or relative (type 2) deficiency of insulin-producing beta cells. The mechanisms governing replication of terminally differentiated beta cells and neogenesis from progenitor cells are unclear. Mice lacking insulin receptor substrate-2 (Irs2) develop beta cell failure, suggesting that insulin signaling is required to maintain an adequate beta cell mass. We report that haploinsufficiency for the forkhead transcription factor Foxo1 reverses beta cell failure in Irs2(-/-) mice through partial restoration of beta cell proliferation and increased expression of the pancreatic transcription factor pancreas/duodenum homeobox gene-1 (Pdx1). Foxo1 and Pdx1 exhibit mutually exclusive patterns of nuclear localization in beta cells, and constitutive nuclear expression of a mutant Foxo1 is associated with lack of Pdx1 expression. We show that Foxo1 acts as a repressor of Foxa2-dependent (Hnf-3beta-dependent) expression from the Pdx1 promoter. We propose that insulin/IGFs regulate beta cell proliferation by relieving Foxo1 inhibition of Pdx1 expression in a subset of cells embedded within pancreatic ducts.
Journal of Clinical Investigation 01/2003; 110(12):1839-47. · 15.39 Impact Factor
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ABSTRACT: Type 2 diabetes results from impaired action and secretion of insulin. It is not known whether the two defects share a common pathogenesis. We show that haploinsufficiency of the Foxo1 gene, encoding a forkhead transcription factor (forkhead box transcription factor O1), restores insulin sensitivity and rescues the diabetic phenotype in insulin-resistant mice by reducing hepatic expression of glucogenetic genes and increasing adipocyte expression of insulin-sensitizing genes. Conversely, a gain-of-function Foxo1 mutation targeted to liver and pancreatic beta-cells results in diabetes arising from a combination of increased hepatic glucose production and impaired beta-cell compensation due to decreased Pdx1 expression. These data indicate that Foxo1 is a negative regulator of insulin sensitivity in liver, adipocytes and pancreatic beta-cells. Impaired insulin signaling to Foxo1 provides a unifying mechanism for the common metabolic abnormalities of type 2 diabetes.NOTE: In the AOP version of this article, the name of the fourth author was misspelled as W K Cavanee rather than the correct spelling: W K Cavenee. This has been corrected in the full-text online version of the article. The name will appear correctly in the print version.
Nature Genetics 11/2002; 32(2):245-53. · 35.53 Impact Factor
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ABSTRACT: The A33 antigen is a transmembrane protein expressed almost exclusively by intestinal epithelial cells. The level of its expression is robust and uniform throughout the rostrocaudal axis of the human and mouse intestines. In the colon, strong expression is found in the basolateral membranes of both the proliferating cells in the lower regions of the crypts and the differentiating cells in the upper regions of crypts. Similarly, in the small intestine, the protein is highly expressed by all the epithelial cells in the crypts and by the differentiated cells migrating over the villi. Thus, the A33 antigen has emerged as a definitive marker for all intestinal epithelial cells, irrespective of cell lineage and differentiation status. To understand the molecular mechanisms mediating this rare tissue-specific expression pattern, we undertook a comprehensive analysis of the 5'-regulatory region of the human A33 antigen gene. This allowed us to point to positive cis-regulatory elements incorporating consensus Krüppel-like factor and caudal-related homeobox (CDX)-binding sites, located just upstream from the human A33 antigen transcription start site, as being important for the intestine-specific expression pattern of this gene. Further analysis provided evidence that the A33 antigen gene may be one of only a few target genes to be described thus far for the intestine-specific homeobox transcription factor, CDX1. Taken together, our data lead us to propose that the activity of CDX1 is pivotal in mediating the exquisite, intestine-specific expression pattern of the A33 antigen gene.
Journal of Biological Chemistry 10/2002; 277(37):34531-9. · 4.77 Impact Factor
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Archives of Biochemistry and Biophysics 08/2002; 403(2):292-8. · 2.93 Impact Factor
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