The Crystal Structure of Abl Kinase with BMS-354825, a Dual SRC/ABL Kinase Inhibitor.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 553-553 ◽  
Author(s):  
John S. Tokarski ◽  
John Newitt ◽  
Francis Y. Lee ◽  
Louis Lombardo ◽  
Robert Borzilleri ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Small Molecule ◽  
Kinase Inhibitor ◽  
High Rate ◽  
Dimensional Structure ◽  
Dual Function ◽  
Activation Loop ◽  
Imatinib Resistance ◽  
Abl Kinase ◽  
Imatinib Resistant

Abstract Chronic myeloid leukemia (CML) is a stem cell disorder caused by a constitutively activated tyrosine kinase, the BCR-ABL oncoprotein. Imatinib (STI571, Gleevec) is a small-molecule inhibitor of this kinase that produces clinical remissions in CML patients and is now frontline therapy for this disease. While this agent has a high rate of clinical success in early phases of CML, development of resistance to this drug becomes increasingly problematic in later stages of the disease. BMS-354825, a small-molecule dual-function SRC/ABL tyrosine kinase inhibitor, appears to overcome many of the limitations associated with imatinib therapy. BMS-354825 is 500-fold more potent than imatinib against BCR-ABL and more importantly retains activity against 14 of 15 imatinib-resistant BCR-ABL mutants (Shah et al., Science, 2004;305(5682):399). In addition, BMS-354825 proved to be equally effective against several pre-clinically and clinically derived tumor models of imatinib resistance (Lee et al., Proceedings of the AACR, Volume 45, March 2004 abstract number 3937). In order to better understand the molecular basis of the relationship between inhibitor chemistry and biological activity, the three-dimensional structure of the kinase domain of Abl kinase complexed with BMS-354825 was determined by X-ray crystallography. The structure reveals that BMS-354825 binds in the ATP-binding site. A comparison with the imatinib-Abl complex (PDB entry 1IEP) reveals that the central cores of BMS-354825 and imatinib occupy overlapping regions but that these two inhibitors extend in opposite directions. The activation loop is observed to be in the active conformation in the presence of bound BMS-354825 in contrast to bound imatinib. There do not appear to be any steric clashes that would preclude BMS-354825 from also binding to the inactive conformation of the activation loop. This observation suggests that the increased binding affinity of BMS-354825 over imatinib is at least partially due to its apparent ability to recognize multiple states of the enzyme. The P-loop is partially disordered as indicated by high B-factors and broken electron density which suggests that interactions between this part of the protein and BMS-354825 are less critical for binding. Interestingly, several imatinib-resistant mutations occur in the P-loop. The structure was analyzed for the 15 imatinib-resistant BCR-ABL mutants and attempts are made to rationalize the activity of BMS-354825 against these mutants.

BMS-354825, a Dual SRC/ABL Kinase Inhibitor, Displays Potent Anti-Tumor Activity in a Model of Intracranial CML Growth.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1988-1988 ◽  
Author(s):  
Robert Wild ◽  
Stephen Castaneda ◽  
Christine Flefleh ◽  
Krista Fager ◽  
Ivan Inigo ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Blood Brain Barrier ◽  
Small Molecule ◽  
Kinase Inhibitor ◽  
High Rate ◽  
Brain Barrier ◽  
Preclinical Model ◽  
Dual Function ◽  
Imatinib Resistance ◽  
Blood Brain

Abstract Chronic myeloid leukemia (CML) is a stem cell disorder caused by a constitutively activated tyrosine kinase, the BCR-ABL oncoprotein. Imatinib (STI571, Gleevec) is a small-molecule inhibitor of this kinase that produces clinical remissions in CML patients and is now frontline therapy for this disease. While this agent has a high rate of clinical success in early phases of CML, development of resistance to this drug is increasingly becoming problematic, particularly in later stages of the disease. Moreover, growing evidence suggests that imatinib has very poor penetration of the blood brain barrier, likely due at least partly to its being a substrate of P-glycoprotin (Pgp), resulting in subtherapeutic levels in the CNS. As a result, several clinical cases have been reported where CNS relapses occurred in imatinib treated CML patients despite peripheral blood and bone marrow complete responses (Leis et al., Leuk Lymphoma. 2004 Apr;45(4):695–8). This phenomenon has also been recapitulated in at least one preclinical model, where the limited ability of imatinib to cross the blood-brain barrier allowed the CNS to become a sanctuary for BCR-ABL-induced leukemia (Wolff et al., Blood. 2003 Jun 15;101(12):5010–3). BMS-354825, a small-molecule dual-function SRC/ABL tyrosine kinase inhibitor, was designed to overcome many of the limitations associated with imatinib therapy. BMS-354825 has more than 500-fold increased potency relative to imatinib versus BCR-ABL and more importantly retains activity against 14 of 15 imatinib-resistant BCR-ABL mutants (Shah et al., Science, 2004 Jul 16;305(5682):399–401). In addition, BMS-354825 proved to be equally effective against several preclinically- and clinically-derived tumor models of imatinib resistance (Lee et al., Proceedings of the AACR, Volume 45, March 2004). In the current study, we assessed the efficacy of BMS-354825, which is not a Pgp substrate, in a model of established intracranial CML tumors. SCID-beige mice bearing K562 CML tumors implanted intracranially (2x106 cells per animal) were treated with BMS-354825 orally b.i.d. for a period of up to 40 days. BMS-354825 proved to be exceptionally efficacious resulting in increased lifespan of animals by 450% and 268% for the 15 mg/kg and 5 mg/kg dose levels, respectively. In order to more directly assess the anti-tumor activities of BMS-354825 in this intracranial CML model, we implanted K562 cells stably transfected with the firefly luciferase gene intracranially into SCID-beige animals. Bioluminescent imaging (BLI) then allowed the non-invasive monitoring of in vivo growth of these tumors. BMS-354825 at 15 mg/kg (2qdx14;6 po) achieved tumor regressions and subsequent complete stasis of intracranial K562 growth while animals were on therapy. In summary, these results suggest that BMS-354825 may have therapeutic advantages over imatinib in the management of intracranial CML disease and warrants further clinical investigation.

The SRC/ABL Inhibitor BMS-354825 Overcomes Resistance to Imatinib Mesylate in Chronic Myelogenous Leukemia Cells through Multiple Mechanisms.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1989-1989 ◽  
Author(s):  
Nicholas J. Donato ◽  
Ji Wu ◽  
Ling Y. Kong ◽  
Francis Lee ◽  
Moshe Talpaz
Keyword(s):  
Tyrosine Kinase ◽  
Tyrosine Kinase Inhibitor ◽  
Kinase Inhibitor ◽  
Myelogenous Leukemia ◽  
Imatinib Resistance ◽  
Abl Kinase ◽  
Lyn Kinase ◽  
Abl Kinases ◽  
Imatinib Resistant ◽  
Dual Inhibition

Abstract BCR-ABL is an oncogenic tyrosine kinase expressed in chronic myelogenous leukemia (CML) cells and is the main target of the tyrosine kinase inhibitor imatinib mesylate. Imatinib-based CML therapy induces hematological and cytogenetic remission in early phase CML patients whereas more advanced patients frequently develop resistance to imatinib by multiple mechanisms, including mutations in the BCR-ABL kinase domain and over-expression of tyrosine kinases that are not inhibited by imatinib. These observations suggest that dual inhibition of src and abl kinases may circumvent imatinib resistance and provide more effective therapy for CML. BMS-354825 is a novel tyrosine kinase inhibitor that inhibits both abl and src kinases at low nM concentrations and is currently being clinically evaluated in imatinib resistant or intolerant CML patients. Our earlier studies demonstrated that increased expression of the src-related kinase Lyn in BCR-ABL expressing K562 cells was associated with imatinib resistance in this cell model and some CML patients. To determine whether inhibition of SRC/ABL kinases differentially affects imatinib sensitive K562 (BCR-ABL +, Lyn −) and resistant K562R (BCR-ABL +, Lyn +) cells were treated with imatinib or BMS-354825 before analysis of cell growth, survival and signaling. BMS-354825 induced apoptosis in both K562 and K562R cells which correlated with inhibition of both Lyn activation and BCR-ABL signaling (CrkL). BMS-354825 effectively reduced both K562 and K562R tumor growth in nude mice whereas imatinib had minimal effects on K562R tumors. Clinical specimens from imatinib resistant CML patients (with and without BCR-ABL kinase mutations) were treated with imatinib or BMS-354825 and analyzed for changes in Lyn and Hck activation. While imatinib had minimal inhibitory effects on Lyn/Hck activation, BMS-354825 completely suppressed Lyn/Hck phosphorylation which correlated with its greater anti-tumor activity in CML samples. BCR-ABL tyrosine phosphorylation was not inhibited by imatinib in Cos cells co-expressing BCR-ABL and Lyn kinase and loss of imatinib sensitivity was totally dependent on Lyn kinase activity. BMS-354825 reduced both Lyn and BCR-ABL activation in co-expressing cells, suggesting that Lyn-mediated phosphorylation plays a direct role in imatinib resistance. We conclude that dual inhibition of SRC/ABL kinases in CML cells by BMS-354825 overcomes resistance to imatinib in vitro and in vivo and induces anti-tumor effects in CML patient specimens resistant to imatinib through expression of imatinib-inactivating BCR-ABL kinase mutations as well as other resistance mechanisms.

Lyn Kinase Alters Gab2 Phosphorylation and c-Cbl Protein Levels To Regulate Imatinib Sensitivity and Survival of Chronic Myelogenous Leukemia Cells.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 2132-2132
Author(s):  
Ji Wu ◽  
Feng Meng ◽  
Moshe Talpaz ◽  
Nicholas J. Donato
Keyword(s):  
Tyrosine Phosphorylation ◽  
Kinase Inhibitor ◽  
K562 Cells ◽  
Imatinib Resistance ◽  
Kinase Inhibition ◽  
Protein Levels ◽  
Abl Kinase ◽  
Lyn Kinase ◽  
Imatinib Resistant ◽  
Cbl Protein

Abstract The tyrosine kinase inhibitor imatinib mesylate (Gleevec) is effective in controlling BCR-ABL expressing leukemias but resistance occurs in some early phase patients while it is more common in advanced disease. Resistance has been generally associated with mutations in the BCR-ABL kinase that effect drug affinity. However patients are also increasingly reported to fail imatinib therapy while retaining wild-type BCR-ABL expression. Our previous studies suggested a role for Lyn, a Src-related kinase, in imatinib resistance. K562 cells selected for imatinib resistance (K562R) overexpress Lyn kinase and its targeted silencing overcomes imatinib resistance and engages apoptosis. Overexpression of Lyn in K562 cells reduces imatinib sensitivity (3-fold) and patients that fail imatinib therapy in the absence of BCR-ABL mutations express a highly activated Lyn kinase that is not suppressed by imatinib. Silencing Lyn expression in patient specimens induces changes in cell survival that are proportional to the level of Lyn protein reduction. To understand the role of Lyn kinase in imatinib resistance and apoptosis we examined proteins associated with this kinase in imatinib resistant cell lines, leukemic cells overexpressing Lyn and specimens derived from imatinib resistant patients. Lyn overexpression blocked complete suppression of BCR-ABL tyrosine phosphorylation by imatinib and affected BCR-ABL signaling adaptors. Although BCR-ABL forms a stable complex with the leukemogenic-critical adaptor protein Gab2 in imatinib sensitive cells, Lyn overexpression resulted in the formation of Lyn:Gab2 complexed in resistant cells. BCR-ABL kinase inhibition failed to reduce tyrosine phosphorylation of Gab2 in these cells while Lyn silencing or kinase inhibition (with dasatinib) completely suppressed Gab2 tyrosine phosphorylation and correlated with the induction of apoptosis. Lyn silencing in K562R cells also lead to a reciprocal increase in the tyrosine phosphorylation and association with a protein of ~120kDa, identified as the E3 ligase, c-Cbl. Lyn overexpression in K562 cells reduced their imatinib sensitivity and reduced c-Cbl protein levels. Kinase inhibitor and co-transfection studies demonstrated that tyrosine phosphorylation of c-Cbl at a critical signaling site (Y774) is primarily controlled by BCR-ABL and deletion or mutation of the c-Cbl RING domain altered its BCR-ABL phosphorylation. These results suggest that c-Cbl complexes are regulated at both the protein and phosphorylation level by Lyn and BCR-ABL kinase activities, respectively. Overexpression and/or activation of Lyn may disrupt the balance and regulation of critical regulators of leukemogenic signaling (Gab2) or protein trafficking and stability (c-Cbl), resulting in increased cell survival and reduced responsiveness to BCR-ABL kinase inhibition. We conclude that Lyn alters the level and function of critical signaling adaptor proteins in CML cells.

Philadelphia-positive patients who already harbor imatinib-resistant Bcr-Abl kinase domain mutations have a higher likelihood of developing additional mutations associated with resistance to second- or third-line tyrosine kinase inhibitors

Blood ◽  
2009 ◽  
Vol 114 (10) ◽  
pp. 2168-2171 ◽  
Author(s):  
Simona Soverini ◽  
Alessandra Gnani ◽  
Sabrina Colarossi ◽  
Fausto Castagnetti ◽  
Elisabetta Abruzzese ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Tyrosine Kinase Inhibitors ◽  
Kinase Inhibitors ◽  
Sequential Therapy ◽  
Initial Response ◽  
Kinase Domain ◽  
Imatinib Resistance ◽  
Mutation Status ◽  
Abl Kinase ◽  
Imatinib Resistant

Abstract Dasatinib and nilotinib are tyrosine kinase inhibitors (TKIs) developed to overcome imatinib resistance in Philadelphia-positive leukemias. To assess how Bcr-Abl kinase domain mutation status evolves during sequential therapy with these TKIs and which mutations may further develop and impair their efficacy, we monitored the mutation status of 95 imatinib-resistant patients before and during treatment with dasatinib and/or nilotinib as second or third TKI. We found that 83% of cases of relapse after an initial response are associated with emergence of newly acquired mutations. However, the spectra of mutants conferring resistance to dasatinib or nilotinib are small and nonoverlapping, except for T315I. Patients already harboring mutations had higher likelihood of relapse associated with development of further mutations compared with patients who did not harbor mutations (23 of 51 vs 8 of 44, respectively, for patients who relapsed on second TKI; 13 of 20 vs 1 of 6, respectively, for patients who relapsed on third TKI).

Variable sensitivity of FLT3 activation loop mutations to the small molecule tyrosine kinase inhibitor MLN518

Blood ◽  
2004 ◽  
Vol 104 (9) ◽  
pp. 2867-2872 ◽  
Author(s):  
Jennifer J. Clark ◽  
Jan Cools ◽  
David P. Curley ◽  
Jin-Chen Yu ◽  
Nathalie A. Lokker ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Small Molecule ◽  
Kinase Inhibitor ◽  
Relative Sensitivity ◽  
Activation Loop ◽  
Kinase Activation ◽  
Downstream Effectors ◽  
Biochemical Assays ◽  
Flt3 Inhibitor ◽  
Variable Sensitivity

Abstract FLT3 is constitutively activated by internal tandem duplications (ITDs) in the juxtamembrane domain or by activation loop mutations in acute myeloid leukemia (AML). We tested the sensitivity of 8 activation loop mutations to the small molecule FLT3 inhibitor, MLN518. Each FLT3 activation loop mutant, including D835Y, D835A, D835E, D835H, D835N, D835V, D835del, and I836del, transformed Ba/F3 cells to factor-independent proliferation and had constitutive tyrosine kinase activation, as assessed by FLT3 autophosphorylation and activation of downstream effectors, including STAT5 and ERK. MLN518 inhibited FLT3 autophosphorylation and phosphorylation of STAT5 and ERK in FLT3-ITD-transformed Ba/F3 cells with an IC50 (50% inhibition of cell viability) of approximately 500 nM. However, there was a broad spectrum of sensitivity among the 8 activation loop mutants, with IC50 ranging from approximately 500 nM to more than 10 μM for the inhibition of phosphorylation of FLT3, STAT5, and ERK. The relative sensitivity of the mutants to MLN518 in biochemical assays correlated with the cellular IC50 for cytokine-independent proliferation of FLT3-transformed Ba/F3 cells in the presence of MLN518. Thus, certain activation loop mutations in FLT3 simultaneously confer resistance to small molecule inhibitors. These findings have implications for the evaluation of responses in clinical trials with FLT3 inhibitors and provide a strategy to screen for compounds that can overcome resistance.

Hematologic and Cytogenetic Responses in Imatinib-Resistant Chronic Phase Chronic Myeloid Leukemia Patients Treated with the Dual SRC/ABL Kinase Inhibitor BMS-354825: Results from a Phase I Dose Escalation Study.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1-1 ◽  
Author(s):  
Charles L. Sawyers ◽  
Neil P. Shah ◽  
Hagop M. Kantarjian ◽  
Nicholas Donato ◽  
John Nicoll ◽  
...  
Keyword(s):  
Phase I ◽  
Dose Escalation ◽  
Clinical Benefit ◽  
Kinase Inhibitor ◽  
Chronic Phase ◽  
Pharmacokinetic Studies ◽  
Imatinib Resistance ◽  
Abl Kinase ◽  
Imatinib Resistant

Abstract Disease relapse in CML patients treated with imatinib is often associated with mutations in the BCR-ABL gene that interfere with the ability of imatinib to block BCR-ABL kinase activity. BMS-354825 is a novel, orally available, dual SRC/ABL kinase inhibitor with more than 100-fold greater potency than imatinib that has in vitro and in vivo preclinical activity against 14 of 15 imatinib resistant BCR-ABL mutants (Shah et al, Science, 305:399, 2004). Here we report the phase I clinical results of BMS-354825 in Philadelphia chromosome positive (Ph+) CML patients in chronic phase with hematologic progression or intolerance while being treated with imatinib. To date (Aug 6, 2004), 29 patients have been treated on 9 cohorts with doses ranging from 15 to 180 mg of BMS-354825 per day given in single or divided doses for 5–7 days per week, for up to 9 months. Similar to imatinib, BMS-354825 has been well tolerated in all patients, with a single episode of grade 4 thrombocytopenia as the only potential drug related adverse event. BMS-354825 is rapidly absorbed with peak concentrations achieved within 2 hours and a terminal phase half-life of about 5 hours. Serum levels well above the concentration required to block CML cell proliferation in vitro have been readily achieved without side effects. Pharmacodynamic studies demonstrate greater than 50 percent inhibition of phosphorylation of the BCR-ABL substrate CRKL and the SRC kinase Lyn, consistent with the serum concentrations observed in pharmacokinetic studies. Patients not receiving optimal clinical benefit were escalated to the next higher dose for which safety parameters were available, thereby allowing a preliminary assessment of clinical activity. To date, 26 patients (22 with imatinib resistance, 4 with imatinib intolerance; average CML duration 6.1 years) have been followed for greater than 4 weeks and are eligible for assessment of hematologic benefit. 22 patients had detectable BCR-ABL kinase domain mutations prior to starting BMS-354825. All 26 patients have been treated with doses of 35 mg per day or greater and have had clinical benefit, including 19 with complete hematologic responses (73%). Of the 7 partial responders, two subsequently had disease progression, one of whom had expansion of a CML subclone containing the imatinib-resistant T315I mutation in BCR-ABL, which also confers resistance to BMS-354825 in preclinical studies. The other 5 partial responders are now being treated with higher doses to attempt conversion to complete hematologic response. 11 of 21 patients (52%) treated for greater than 3 months have cytogenetic benefit, including 6 major (1–35% Ph+), 1 minor (36–65% Ph+) and 4 minimal (66–95% Ph+) cytogenetic responses. One patient has achieved complete cytogenetic response. Dose escalation continues, and phase II studies in chronic, accelerated and blast crisis CML are currently being initiated. These data provide compelling evidence supporting the safety and efficacy of BMS-354825 in imatinib-resistant chronic phase CML.

Comparative Analysis of Two BCR-ABL Small Molecule Inhibitors Reveals Overlapping but Distinct Mechanisms of Resistance.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 552-552 ◽  
Author(s):  
Michael R. Burgess ◽  
Neil P. Shah ◽  
Brian J. Skaggs ◽  
Francis Y. Lee ◽  
Charles L. Sawyers
Keyword(s):  
Small Molecule ◽  
Small Molecule Inhibitors ◽  
Point Mutations ◽  
Kinase Domain ◽  
Drug Binding ◽  
Saturation Mutagenesis ◽  
Imatinib Resistance ◽  
Abl Kinase ◽  
Imatinib Resistant

Abstract A novel dual SRC/ABL kinase inhibitor, BMS-354825, is showing promise for the treatment of imatinib-resistant chronic myeloid leukemia not only in vitro (Shah NP, et al., Science 305:399), but also in a phase I clinical trial (ASH abstract: Sawyers CL, et al.) Resistance to imatinib is increasingly found in patients due to point mutations in the BCR-ABL kinase domain that do not impair kinase activity but prevent drug binding. BMS-354825 is more potent than imatinib and retains activity against 14 of 15 imatinib-resistant BCR-ABL mutants in vitro. The compound’s ability to inhibit imatinib-resistant forms of BCR-ABL is presumed to be due to its relaxed binding requirements, whereas imatinib requires the adoption of a closed conformation of the kinase to bind. We addressed the hypothesis that the relaxed binding requirements of BMS-354825 would limit the range of BCR-ABL mutations that confer drug resistance. To address this question, we employed a saturation mutagenesis experiment as described by others (Azam M, et al., Cell 112:831) and found that the spectrum of BMS-354825-resistant mutants was reduced compared to that of imatinib. In a series of such screens, mutations at only four amino acids have been isolated, two of which account for the vast majority of resistant clones. In contrast, Azam et al. isolated over 20 mutations in a screen for imatinib resistance, a finding which has been generally reproduced in our lab. All four BMS-354825-resistant mutations map to known BMS-354825 contact residues as shown by co-crystallographic studies (ASH abstract: Tokarski JS et al., Bristol-Myers Squibb). Mutations at L248, T315, and F317 show BMS-354825 resistance and have been previously reported to confer imatinib resistance. Mutation at V299 represents a novel mode of resistance. Interestingly, some point mutations conferring BMS-354825 resistance were at positions known to be mutated in cases of imatinib resistance, but the mutated residues differed. Furthermore, the identity of the mutated residue was crucial in conferring sensitivity or resistance to an individual drug as shown by comparison of cellular IC50’s (see table). For example, F317L was shown previously to confer imatinib resistance. F317V, on the other hand, demonstrates relative BMS-354825-resistance but is still exquisitely sensitive to imatinib. In a screen for mutants simultaneously resistant to both drugs, we consistently recover 30–50 fold fewer mutant clones compared to single drug treatment. All such clones isolated to date encode for T315I. Kinase domain point mutation is becoming an increasingly encountered clinical problem in diseases treated with small molecule inhibitors. Our findings suggest that combination therapy with imatinib and BMS-354825 may be of clinical utility in CML, particularly by delaying the development of resistance. IC50 for growth (nM) Baf3 Clone imatinib BMS-354825 p210 wt < 1,000 < 5 T315I > 10,000 > 500 T315A 1,000 100 F317L 2,000 10 F317V < 1,000 60 V299L 1,000 20 L248R > 10,000 20

Imatinib-Resistant BCR-ABL Mutant Cellular Screening Paradigm To Characterize Promising Small-Molecule Inhibitors: T315I-BCR-ABL Targeted Drug Discovery.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 2181-2181
Author(s):  
Mohammad Azam ◽  
William C. Shakespeare ◽  
Chester Metcalf ◽  
Yihan Wang ◽  
Raji Sunderamoorthi ◽  
...  
Keyword(s):  
Drug Discovery ◽  
Structural Analysis ◽  
Cell Lines ◽  
Small Molecule ◽  
Kinase Inhibitors ◽  
Kinase Inhibitor ◽  
Drug Binding ◽  
Imatinib Resistance ◽  
Inhibition Of Proliferation ◽  
Imatinib Resistant

Abstract In patients with chronic myeloid leukemia (CML), kinase domain mutations account for imatinib resistance in the majority of cases. Mutations cause either a direct steric hindrance to drug binding or a conformational change that favors kinase activation, which therefore precludes imatinib binding. We have previously characterized the dual Src-Abl kinase inhibitor AP23464 and found it to effectively suppress the growth of cells expressing native and essentially all imatinib-resistant variants of BCR-ABL, with the notable exception of the gatekeeper T315I mutant (Azam et al., Proc. Natl. Acad. Sci. USA, 103: 9244, 2006). Following this work, we have used mutant panel screening and integrated structural analysis to further characterize key analogs designed to overcome T315I resistance, as exemplified by AP23846 and AP24163. Both molecules effectively inhibit the tyrosine kinase activity of wild type (WT) and T315I variants of BCR-ABL, and inhibit the proliferation of BaF3-derived cell lines expressing these enzymes (see Table below). AP24163 was further characterized against a broader panel of imatinib-resistant BCR-ABL-expressing cell lines and showed a promising profile of proliferation inhibition. Comparison of these data with structural models of the mutants provides insights into the basis for the ability of AP24163 to overcome imatinib resistance. Refinement of small-molecule kinase inhibitors by the integration of sequential screening of panels of mutants coupled with structural analysis is a powerful drug discovery paradigm that is applicable to an increasing number of targeted therapeutic agents. INHIBITION OF PROLIFERATION OF BAF3 CELLS EXPRESSING BCR-ABL AND ITS VARIANTS (IC50 in nM) IMATINIB AP23464 AP23846 AP24163 WT 600 14 500 7 T315I >20000 >1000 500 480 L248R >20000 92 ND 64 G250E 5000 25 ND 63 Q252H 3000 40 ND 42 Y253H 18000 32 ND 44 E255K 12000 74 ND 24 BAF3+IL3 >20000 >1000 500 >10000 Figure Figure

scholarly journals Uniform sensitivity of FLT3 activation loop mutants to the tyrosine kinase inhibitor midostaurin

Blood ◽  
2007 ◽  
Vol 110 (13) ◽  
pp. 4476-4479 ◽  
Author(s):  
Elly V. Barry ◽  
Jennifer J. Clark ◽  
Jan Cools ◽  
Johannes Roesel ◽  
D. Gary Gilliland
Keyword(s):  
Tyrosine Kinase ◽  
Small Molecule ◽  
Small Molecule Inhibitors ◽  
Kinase Inhibitor ◽  
Activation Loop ◽  
Stimuli Responsive ◽  
Activating Mutations ◽  
Extracellular Stimuli ◽  
Tandem Duplications ◽  
Target Effects

Small molecule inhibitors that target fms-like tyrosine kinase 3 (FLT3)–activating mutations have potential in the treatment of leukemias. However, certain mutations can simultaneously activate the tyrosine kinase, and confer resistance to small molecule inhibitors. We therefore tested the sensitivity of 8 FLT3 activation loop mutants to midostaurin. Each mutant conferred IL-3 factor–independent proliferation to Ba/F3 cells, and each resulted in the constitutive activation of FLT3 and its targets, signal transducer and activator of transcription 5 (STAT5) and extracellular stimuli-responsive kinase (ERK). For each mutant tested, midostaurin inhibited cell growth and phosphorylation of FLT3, STAT5, and ERK. In contrast, midostaruin did not inhibit Ba/F3 cells stably transduced with FLT3-internal tandem duplications containing a G697R mutation that confers resistance to midostaurin, demonstrating that midostaurin inhibition of FLT3 activation loop mutants was not due to off-target effects. We conclude that midostaurin is a potent inhibitor of a spectrum of FLT3 activation loop mutations, and that acute myeloid leukemia patients with such mutations are potential candidates for clinical trials involving midostaurin.

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