A Single Amino Acid Exchange Inverts Susceptibility of Related Receptor Tyrosine Kinases for the ATP Site Inhibitor STI-571

Abstract

The tyrosine kinase inhibitor STI-571 potently blocks BCR-Abl, platelet-derived growth factor (PDGF) ␣-and ␤-receptors, and c-Kit kinase activity. Flt3, a receptor tyrosine kinase closely related to PDGF receptors and c-Kit is, however, not inhibited by STI-571. Sequence alignments of different kinases and indications from the crystal structure of the STI-571 Abl kinase complex revealed amino acid residues that are probably crucial for this activity profile. It was predicted that Flt3 Phe-691 in the ␤5 strand may sterically prevent interaction with STI-571. The point mutants Flt3 F691T and PDGF␤-receptor T681F were constructed, and kinase assays showed that the Flt3 mutant but not the PDGF␤-recep- PDGFR-␣ and -␤ are members of the class III receptor tyrosine kinases. Aberrant activation of PDGF receptors has been linked to several disease states including certain malignancies and atherosclerosis, restenosis, and fibrotic conditions (7). Selective PDGF receptor tyrosine kinase inhibitors have therefore been developed. These include phenylaminopyrimidines (8) such as STI-571, phenylbenzimidazoles (9, 10), quinoxalines (11, 12), 6,7-dimethoxyquinolines (13), and bis(1H-2-indolyl) methanones (14).Flt3 (Flk2, STK1) is structurally closely related to the PDGF receptor kinases and c-Kit. It is overexpressed in various types of leukemia, including B-lineage acute lymphoblastic leukemia and acute myeloid leukemia as well as T-lineage acute lymphoblastic leukemia and chronic myelogenous leukemia blast crisis cells (15)(16)(17). Different activating mutations in the Flt3 gene have been detected in acute myeloid leukemia patients. Flt3 may, therefore, be a suitable target for therapy of Flt3-dependent leukemias. Despite its close homology to PDGF receptors, Flt3 kinase is not inhibited by STI-571.Multiple sequence alignments and the three-dimensional structure of the Abl kinase STI-571 complex (6) indicate possible reasons for selectivity. In some chronic myelogenous leukemia patients who responded initially to STI-571 but then relapsed, the resistance to the drug was associated with a single T315I mutation in the ␤5 strand of the Abl kinase domain (18). The side chain of Thr-315 is assumed to form a critical O-HN hydrogen bond with the pyrimidinylamino group of STI-571. Replacement of Abl Thr-315 by IRK Met-1076 has been sug-* This work was supported in part by grants from Deutsche Krebshilfe, e.V. (10-1717-Do I to S. D., F. D. B., and S. M.), the Deutsche Forschungsgemeinschaft (Se 600/2-4), and Interdisziplinä res Zentrum fü r Klinische Forschung and Fonds Innovative Medizinische Forschung Mü nster (to H. S.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.□ S The on-line version of this article (available at

Inhibition of RET tyrosine kinase by SU5416

“…A similar phenomenon was observed for EGFR, FGFR, and PDGFR (Blencke et al 2004). On the other hand, changing Phe-691 to smaller threonine rendered FLT-3 kinase sensitive to Imatinib (Bohmer et al 2003). In contrast, three indolinone compounds were found to be insensitive to mutations of this particular residue, suggesting that this class of inhibitors may represent an exception to the rule (Blencke et al 2004).…”

FLT3: ITDoes matter in leukemia

“…However, substitution of Phe-691 with threonine renders the receptor susceptible to the drug (IC 50 0.1-0.3 mM). 148 FLT3 inhibitors cause cell cycle arrest, inhibit proliferation, and induce apoptosis in a variety of FLT3-dependent cell lines. In a number of different studies, the FLT3 inhibitors displayed in Table 5 have been shown to be cytotoxic to the human leukemia-derived cell lines MV4-11, MOLM-13, MOLM-14, EOL-1, and SEMK2, all of which express constitutively activated FLT3.…”

“…Even though these compounds are all likely to fit into the ATP binding pocket of the receptor, some may do so via an induced fit mechanism (like the indolocarbazole staurosporine appears to do when binding to CSK), while others bind in a lock-and-key manner (such as imatinib binding to ABL). 147,148 Inferring from the situation with other drugs, FLT3 inhibitors may bind to the active, inactive, or transitional state of FLT3. Selectivity for FLT3 may be greatly influenced by differences in single amino-acid residues between different receptors.…”

Denaturing-HPLC-Based Assay for Detection of ABL Mutations in Chronic Myeloid Leukemia Patients Resistant to Imatinib

“…Structural data suggest that Imatinib acts by blocking the BCR-ABL protein in an inactive conforma-tion (12,13 ), which prevents the transfer of phosphate from ATP to substrates and blocks the downstream signal transduction pathways (14 -16 ). It has been postulated that mutations within the ATP-binding site can prevent Imatinib from binding, either interrupting critical contact points between Imatinib and the protein or inducing a conformation to which Imatinib is unable to bind (17,18 ). Several mutations have been reported in association with the resistant phenotype, and most of them are well characterized in terms of their ability and degree to which they induce resistance (4,7,9,19,20 ).…”