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Supplementary Figure 1 from In vitro Activity of Bcr-Abl Inhibitors AMN107 and BMS-354825 against Clinically Relevant Imatinib-Resistant Abl Kinase Domain Mutants

Supplementary Figure 2 from In vitro Activity of Bcr-Abl Inhibitors AMN107 and BMS-354825 against Clinically Relevant Imatinib-Resistant Abl Kinase Domain Mutants

Imatinib, a Bcr-Abl tyrosine kinase inhibitor, is a highly effective therapy for patients with chronic myelogenous leukemia (CML). Despite durable responses in most chronic phase patients, relapses have been observed and are much more prevalent in patients with advanced disease. The most common mechanism of acquired imatinib resistance has been traced to Bcr-Abl kinase domain mutations with decreased imatinib sensitivity. Thus, alternate Bcr-Abl kinase inhibitors that have activity against imatinib-resistant mutants would be useful for patients who relapse on imatinib therapy. Two such Bcr-Abl inhibitors are currently being evaluated in clinical trials: the improved potency, selective Abl inhibitor AMN107 and the highly potent dual Src/Abl inhibitor BMS-354825. In the current article, we compared imatinib, AMN107, and BMS-354825 in cellular and biochemical assays against a panel of 16 kinase domain mutants representing >90% of clinical isolates. We report that AMN107 and BMS-354825 are 20-fold and 325-fold more potent than imatinib against cells expressing wild-type Bcr-Abl and that similar improvements are maintained for all imatinib-resistant mutants tested, with the exception of T315I. Thus, both inhibitors hold promise for treating imatinib-refractory CML.

Binding of tyrosine kinase inhibitors such as imatinib was shown to induce a novel open-inhibited conformation of BCR-ABL, in which Tyr245 is exposed and prone to phosphorylation. To evaluate whether this leads to priming of the kinase in cellular systems, we probed activation of downstream signaling as a result of Tyr245 phosphorylation in a series of cellular washout experiments. While a spike in Tyr245 phosphorylation was observed both in overexpression and endogenous settings, no induction of downstream signaling was detected, showing that the priming hypothesis is not relevant for the therapeutic situation.

This paper describes the identification of 6-(pyrimidin-4-yloxy)-naphthalene-1-carboxamides as a new class of potent and selective human vascular endothelial growth factor receptor 2 (VEGFR2) tyrosine kinase inhibitors. In biochemical and cellular assays, the compounds exhibit single-digit nanomolar potency toward VEGFR2. Compounds of this series show good exposure in rodents when dosed orally. They potently inhibit VEGF-driven angiogenesis in a chamber model and rodent tumor models at daily doses of less than 3 mg/kg by targeting the tumor vasculature as demonstrated by ELISA for TIE-2 in lysates or by immunohistochemical analysis. This novel series of compounds shows a potential for the treatment of solid tumors and other diseases where angiogenesis plays an important role.

Although orphan drug applications required by the EMEA must include assessments of similarity to pre-existing products, these can be difficult to quantify. Here we illustrate a paradigm in comparing nilotinib to the prototype kinase inhibitor imatinib, and equate the degree of structural similarity to differences in properties. Nilotinib was discovered following re-engineering of imatinib, employing structural biology and medicinal chemistry strategies to optimise cellular potency and selectivity towards BCR-ABL1. Through evolving only to conserve these properties, this resulted in significant structural differences between nilotinib and imatinib, quantified by a Daylight-fingerprint-Tanimoto similarity coefficient of 0.6, with the meaning of this absolute measure being supported by an analysis of similarity distributions of similar drug-like molecules. This dissimilarity is reflected in the drugs having substantially different preclinical pharmacology and a lack of cross-intolerance in CML patients, which translates into nilotinib being an efficacious treatment for CML, with a favourable side-effect profile.

Allosteric inhibitors of Bcr-Abl have emerged as a novel therapeutic option for the treatment of CML. Using fragment-based screening, a search for novel Abl inhibitors that bind to the myristate pocket was carried out. Here we show that not all myristate ligands are functional inhibitors, but that the conformational state of C-terminal helix_I is a structural determinant for functional activity. We present an NMR-based conformational assay to monitor the conformation of this crucial helix_I and show that myristate ligands that bend helix_I are functional antagonists, whereas ligands that bind to the myristate pocket but do not induce this conformational change are kinase agonists. Activation of c-Abl by allosteric agonists has been confirmed in a biochemical assay.

The ATP-competitive inhibitors dasatinib and nilotinib, which bind to catalytically different conformations of the Abl kinase domain, have recently been approved for the treatment of imatinib-resistant CML. These two new drugs, albeit very efficient against most of the imatinib-resistant mutants of Bcr-Abl, fail to effectively suppress the Bcr-Abl activity of the T315I (or gatekeeper) mutation. Generating new ATP site-binding drugs that target the T315I in Abl has been hampered, amongst others, by target selectivity, which is frequently an issue when developing ATP-competitive inhibitors. Recently, using an unbiased cellular screening approach, GNF-2, a non-ATP-competitive inhibitor, has been identified that demonstrates cellular activity against Bcr-Abl transformed cells. The exquisite selectivity of GNF-2 is due to the finding that it targets the myristate binding site located near the C-terminus of the Abl kinase domain, as demonstrated by genetic approaches, solution NMR and X-ray crystallography. GNF-2, like myristate, is able to induce and/or stabilize the clamped inactive conformation of Abl analogous to the SH2-Y527 interaction of Src. The molecular mechanism for allosteric inhibition by the GNF-2 inhibitor class, and the combined effects with ATP-competitive inhibitors such as nilotinib and imatinib on wild-type Abl and imatinib-resistant mutants, in particular the T315I gatekeeper mutant, are reviewed.

Controlling aberrant protein kinase activity is a promising strategy for a variety of diseases, particularly cancer. Hence, the development of kinase inhibitors is currently a focal point for pharmaceutical research. In this study we utilize a chip-based reverse phase protein array (RPA) platform for profiling of kinase inhibitors in cell-based assays. In combination with the planar wave-guide technology the assay system has an absolute LOD down to the low zeptomole range. A431 cell lysates were analyzed for the activation state of key effectors in the epidermal growth factor (EGF) and insulin signaling pathways to validate this model for compound screening. A microtiter-plate format for growing, treating, and lysing cells was shown to be suitable for this approach, establishing the value of the technology as a screening tool for characterization of large numbers of kinase inhibitors against a wide variety of cellular signaling pathways. Moreover, the reverse array format allows rapid development of site-specific phosphorylation assays, since in contrast to ELISA type systems only a single antigen-specific antibody is required.

Patients with advanced stages of chronic myeloid leukemia (CML) often manifest imatinib mesylate resistance associated with point mutations in BCR-ABL. AMN107 is a new higher-potency inhibitor of BCR-ABL. To identify mutations in BCR-ABL that could result in resistance to AMN107, a cDNA library of BCR-ABL mutants was introduced into Ba/F3 cells followed by selection in AMN107 (0.125-0.5 μM). A total of 86 individual, drug-resistant colonies were recovered, and the SH3, SH2, and kinase domains of BCR-ABL were sequenced. A total of 46 colonies had single point mutations in BCR-ABL, with a total of 17 different mutations, all within the kinase domain. The other 40 colonies had multiple point mutations and were not analyzed further. Each of the 17 single point mutants were reconstructed by site-directed mutagenesis of native BCR-ABL and found to be approximately 2.5- to 800-fold more resistant to AMN107 than native BCR-ABL. The mutations included 6 known imatinib mesylate–resistant mutations, including T315I, which showed complete resistance to AMN107. Interestingly, most AMN107-resistant mutants were also resistant to imatinib mesylate. These results may predict some of the resistance mutations that will be detected in clinical trials with this kinase inhibitor.

In advanced-phase chronic myeloid leukemia (CML), resistance to imatinib mesylate is associated with point mutations in the BCR-ABL kinase domain. A new generation of potent ABL kinase inhibitors is undergoing clinical evaluation. It is important to generate specific resistance profiles for each of these compounds, which could translate into combinatorial and sequential treatment strategies. Having characterized nilotinib (AMN107) against a large panel of imatinib mesylate–resistant Bcr-Abl mutants, we investigated which mutants might arise under nilotinib therapy using a cell-based resistance screen. In contrast to imatinib mesylate, resistance to nilotinib was associated with a limited spectrum of Bcr-Abl kinase mutations. Among these were mutations affecting the P-loop and T315I. Rarely emerging resistant colonies at a concentration of 400 nM nilotinib exclusively expressed the T315I mutation. With the exception of T315I, all of the mutations that were identified were effectively suppressed when the nilotinib concentration was increased to 2000 nM, which falls within the peak-trough range in plasma levels (3.6-1.7 μM) measured in patients treated with 400 mg twice daily. Our findings suggest that nilotinib might be superior to imatinib mesylate in terms of the development of resistance. However, our study indicates that clinical resistance to nilotinib may be associated with the predominant emergence of T315I.

Chronic myelogenous leukaemia (CML) and Philadelphia chromosome positive (Ph+) acute lymphoblastic leukaemia (ALL) are caused by the BCR-ABL oncogene. Imatinib inhibits the tyrosine kinase activity of the BCR-ABL protein and is an effective, frontline therapy for chronic-phase CML. However, accelerated or blast-crisis phase CML patients and Ph+ ALL patients often relapse due to drug resistance resulting from the emergence of imatinib-resistant point mutations within the BCR-ABL tyrosine kinase domain. This has stimulated the development of new kinase inhibitors that are able to over-ride resistance to imatinib. The novel, selective BCR-ABL inhibitor, AMN107, was designed to fit into the ATP-binding site of the BCR-ABL protein with higher affinity than imatinib. In addition to being more potent than imatinib (IC50

Chronic myelogenous leukemia (CML) is a myeloproliferative disorder characterized at the molecular level by the expression of Bcr-abl, a 210-kDa fusion protein with deregulated tyrosine kinase activity. Encouraged by the clinical validation of Bcr-abl as the target for the treatment of CML by imatinib, we sought to identify pharmacological agents that could target this kinase by a distinct mechanism. We report the discovery of a new class of Bcr-abl inhibitors using an unbiased differential cytotoxicity screen of a combinatorial kinase-directed heterocycle library. Compounds in this class (exemplified by GNF-2) show exclusive antiproliferative activity toward Bcr-abl-transformed cells, with potencies similar to imatinib, while showing no inhibition of the kinase activity of full-length or catalytic domain of c-abl. We propose that this new class of compounds inhibits Bcr-abl kinase activity through an allosteric non-ATP competitive mechanism.

Imatinib, a Bcr-Abl tyrosine kinase inhibitor, is a highly effective therapy for patients with chronic myelogenous leukemia (CML). Despite durable responses in most chronic phase patients, relapses have been observed and are much more prevalent in patients with advanced disease. The most common mechanism of acquired imatinib resistance has been traced to Bcr-Abl kinase domain mutations with decreased imatinib sensitivity. Thus, alternate Bcr-Abl kinase inhibitors that have activity against imatinib-resistant mutants would be useful for patients who relapse on imatinib therapy. Two such Bcr-Abl inhibitors are currently being evaluated in clinical trials: the improved potency, selective Abl inhibitor AMN107 and the highly potent dual Src/Abl inhibitor BMS-354825. In the current article, we compared imatinib, AMN107, and BMS-354825 in cellular and biochemical assays against a panel of 16 kinase domain mutants representing >90% of clinical isolates. We report that AMN107 and BMS-354825 are 20-fold and 325-fold more potent than imatinib against cells expressing wild-type Bcr-Abl and that similar improvements are maintained for all imatinib-resistant mutants tested, with the exception of T315I. Thus, both inhibitors hold promise for treating imatinib-refractory CML.

Recent studies showed that the 40S ribosomal protein S6 kinase (S6K) p70S6K/p85S6K, termed S6K1 (51), is a major effector of cell growth. This conclusion stems from gene deletion studies with Drosophila (39) and with mice (51) as well as recent studies with cell cultures (11). The loss of the Drosophila S6K (dS6K) gene is semilethal, with the few surviving adults having a severely reduced body size. The larvae of such flies exhibit a long developmental delay, consistent with a twofold increase in cell cycle doubling times. The few surviving adults are quite lethargic, living no longer than 2 weeks, and females are sterile. Surprisingly, the reduction in mass is strictly due to a decrease in cell size rather than to a decrease in cell number (39). In mice, removal of this kinase is not lethal, but the mice are approximately 20% smaller at birth (51). Such mice exhibit normal fasting glucose levels but are mildly glucose intolerant due to markedly reduced levels of circulating insulin (42). Reduced insulin levels are caused by a reduction in pancreatic endocrine mass and an impairment of insulin secretion, which can be traced to a selective reduction in β-cell size. Unexpectedly, the effects on body mass and hypoinsulinemia do not appear to be attributable to a reduction in S6 phosphorylation, as this response proved to be largely intact in S6K1-deficient animals (51). However, S6 phosphorylation in such animals was still sensitive to the bacterial macrolide rapamycin (51), which inhibits the mammalian target of rapamycin (mTOR) (1, 7, 16, 48), the upstream S6K1 kinase (4, 8, 18), suggesting the existence of a second S6K. Subsequent searches of expressed sequence tag databases and biochemical studies led to the identification of S6K2, which exhibited overall homology of over 80% with S6K1 in the highly conserved kinase and linker domains (17, 47, 51). In all tissues examined from S6K1-deficient mice, S6K2 transcripts were upregulated (51). From this observation, it was reasoned that S6K1 and S6K2 functions were redundant and that a deletion of the S6K1 gene led to a compensatory increase in the expression of S6K2. In parallel studies, it was demonstrated that rapamycin suppressed the serum-induced translational upregulation of a family of mRNAs which contain a polypyrimidine tract at their 5′ end (5′-terminal oligopyrimidine [5′TOP] mRNAs) (20, 55). These mRNAs largely code for components of the translational apparatus, most notably, ribosomal proteins (37). Earlier studies had shown that the translation of such transcripts is under selective translational control (22) and requires an intact 5′TOP tract (19, 49). In addition, a dominant interfering allele of S6K1 inhibited the mitogen-induced translational upregulation of 5′TOP mRNAs to the same extent as rapamycin, whereas an activated allele of S6K1, which exhibits a substantial degree of rapamycin resistance, largely protected these transcripts from the inhibitory effects of rapamycin (19, 49). Seemingly consistent with these arguments, in embryonic stem (ES) cells from which S6K1 had been homologously deleted by selection with high doses of G418, serum no longer had an effect on the upregulation of 5′TOP mRNAs, nor was there a redistribution of 5′TOP mRNAs from polysomes to nonpolysomes in the presence of rapamycin (24). However, S6 phosphorylation was initially reported to be abolished in these cells (24), despite the fact that it was largely intact in cells and tissues derived from S6K1−/− mice (51). This difference seemed to be resolved in subsequent studies, where S6 phosphorylation was detected in these same S6K1−/− ES cells and S6K2 was present and active (31, 60). Despite these observations, it was again recently reported that S6 phosphorylation was absent from these same cells (53). Furthermore, it was also claimed in the latter study that S6K activation, S6 phosphorylation, and rapamycin had little impact on 5′TOP mRNA translation in PC12 cells (53), although others working with these same cells had reported earlier that rapamycin treatment abolished the selective recruitment of these transcripts from small to large polysomes (44). Obviously, cells lacking both S6K1 and S6K2 would facilitate such studies. Therefore, we set out to delete the S6K2 gene from mice and to determine whether we could generate S6K1−/−/S6K2−/− mice. Here we report on the deletion of the S6K2 gene and the effects of deleting both S6K1 and S6K2 on animal growth and viability as well as on S6 phosphorylation, cell proliferation, and 5′TOP mRNA translation.

Following the paradigm set by STI571, protein tyrosine kinase inhibitors are emerging as a promising class of drugs, capable of modulating intracellular signaling and demonstrating therapeutic potential for the treatment of proliferative diseases. Although the majority of chronic phase CML patients treated with STI571 respond, some patients, especially those with more advanced disease, relapse. This article reviews the reasons for relapse and, in particular, analyses resistance resulting from Bcr-Abl tyrosine kinase domain mutations at the molecular level. Arguments are based upon the structure of the STI571-Abl complex, which is compared to the crystal structures of PD173955-Abl and PD180970-Abl, which bind to the kinase differently. Strategies to potentially circumvent or overcome resistance are discussed.

The PI3K/AKT/mTOR signaling pathway plays a fundamental role in cell proliferation, growth and survival and aberrant activation of this signaling pathway has been shown to drive the progression of malignant tumors.[1] Drugs targeting the pathway at multiple points, such as dual PI3K/mTOR inhibitors appear to have the broadest activity profile to address cancer therapeutic strategies and are currently being explored in numerous clinical studies. Recently, we presented PQR309, a novel, brain-penetrant pan-PI3K/mTOR inhibitor, which entered phase II clinical trials in 2016.[2] Here, we report the lead optimization of PQR530, a potent and brain-penetrant follow-up compound as pan-PI3K/mTORC1/2 inhibitor. The development of a follow-up compound concentrated on the improvement of both, the potency and the selectivity for all targeted kinases, namely the class IA PI3K isoforms as well as mTOR. We present a detailed ligand-based structure-activity relationship study which was obtained by systematic modifications of the hinge region as well as the affinity binding substituents. This study led to the identification of PQR530, a dual pan-PI3K/mTORC1/2 inhibitor showing excellent activities in cellular assays as well as in PI3Kα and mTOR enzymatic binding assays. In A2058 melanoma cells PQR530 inhibited protein kinase B (PKB, pSer473) and ribosomal protein S6 (pS6, pSer235/236) phosphorylation with IC50 values of 0.07 µM. PQR530 showed excellent selectivity over a wide panel of kinases, as well as excellent selectivity versus unrelated receptor enzymes and ion channels. Moreover, PQR530 displayed potency in a panel of 44 cancer cell lines (NTRC OncolinesTM) to prevent cancer cell growth (mean value for GI50 of 426 nM). Oral application of PQR530 to mice resulted in a dose-proportional PK and demonstrated good oral bioavailability and excellent brain penetration.[3] An optimized, robust synthetic route allowed rapid access to multi-gram quantities of PQR530 for pre-clinical development in only 4 steps. In conclusion, PQR530 inhibits all PI3K isoforms and the mammalian target of rapamycin (mTOR) complexes C1/2 potently and selectively, and shows anti-tumor effects in vitro and in vivo. [1] M. P. Wymann, M. Zvelebil, M. Laffargue (2003). Phosphoinositide 3-kinase signalling – which way to target? Trends Pharmacol Sci.; 24, 366-376. [2] V. Cmiljanovic et. al. “PQR309: Structure-Based Design, Synthesis and Biological Evaluation of a Novel, Selective, Dual Pan-PI3K/mTOR Inhibitor” presented at AACR Annual Meeting 2015, April 18-22, Philadelphia, Pennsylvania, USA. [3] P. Hillmann et al. “Pharmacological Characterization of the Selective, Orally Bioavailable, Potent Dual PI3K/mTORC1/2 Inhibitor PQR530” abstract submitted for AACR Annual Meeting 2017, April 1-5, Washington, D. C., USA. Citation Format: Denise Rageot, Florent Beaufils, Anna Melone, Alexander M. Sele, Thomas Bohnacker, Marc Lang, Jürgen Mestan, Petra Hillmann, Paul Hebeisen, Doriano Fabbro, Matthias P. Wymann. Discovery and biological evaluation of PQR530, a highly potent dual pan-PI3K/mTORC1/2 inhibitor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 140. doi:10.1158/1538-7445.AM2017-140

Introduction: The phosphatidylinositol 3-kinase (PI3K) signaling pathway plays a fundamental role in many cellular processes like growth, survival, proliferation, differentiation and motility. In cancers several mutations have been identified that lead to constitutive activation of PI3K. PQR530 is a novel, ATP site directed inhibitor of all PI3K isoforms and the mammalian target of rapamycin (mTOR) complexes C1/2 that is currently in pre-clinical development. PQR530 potently binds to its targets, inhibits cell proliferation and shows excellent selectivity versus related and unrelated kinases [1]. Results: PQR530 inhibits PI3K signaling in stimulated MCF7 cells as detected by PathScan analysis. Excellent tolerability has been found for PQR530 during GLP toxicological testing in rats and dogs. Increase in insulin and blood glucose, a treatable class effect of PI3K inhibitors, has been observed after PQR530 administration to mice. Investigation of mutagenicity and hERG binding resulted in a clean profile. PQR530 exhibited dose-proportional pharmacokinetics (PK) in male C57BL/6J mice. A maximum concentration (Cmax) in plasma and brain was reached after 30 minutes (7.8 μg/ml and 112.6 μg/ml, respectively) indicating that efficacious concentrations were reached in both tissues. The calculated half-life (t1/2) for plasma and brain was approximately 5 hours. PQR530 potently inhibited PI3K signaling in vivo for several hours after administration of a single oral dose of 50 mg/kg. Tumor growth was significantly decreased in SUDHL-6 lymphoma, RIVA lymphoma and OVCAR-3 ovarian cancer mouse xenografts using daily, oral administration. Conclusion: PQR530 is a potent, ATP competitive pan-PI3K and mTORC1/2 inhibitor. The physico-chemical properties of PQR530 result in good oral bioavailability and excellent brain penetration. PQR530 is well tolerated and efficiently inhibits tumor growth in xenograft models. Preclinical data allow for further development of the compound. [1] Rageot D, et al., Discovery and biological evaluation of PQR530, a highly potent dual pan-PI3K/mTORC1/2 inhibitor, abstract submitted for AACR Annual Meeting 2017, April 1-5, Washington, D. C., USA. Citation Format: Petra Hillmann, Denise Rageot, Florent Beaufils, Anna Melone, Alexander Sele, Robert A. Ettlin, Jürgen Mestan, Vladimir Cmiljanovic, Marc Lang, Elisabeth Singer, Carolin Walter, Hoa HP Nguyen, Paul Hebeisen, Matthias P. Wymann, Doriano Fabbro. Pharmacological characterization of the selective, orally bioavailable, potent dual PI3K/mTORC1/2 inhibitor PQR530 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 159. doi:10.1158/1538-7445.AM2017-159

Skp2 is a member of the F-box family of substrate-recognition subunits of SCF ubiquitin–protein ligase complexes that has been implicated in the ubiquitin-mediated degradation of several key regulators of mammalian G 1 progression, including the cyclin-dependent kinase inhibitor p27, a dosage-dependent tumor suppressor protein. In this study, we examined Skp2 and p27 protein expression by immunohistochemistry in normal oral epithelium and in different stages of malignant oral cancer progression, including dysplasia and oral squamous cell carcinoma. We found that increased levels of Skp2 protein are associated with reduced p27 in a subset of oral epithelial dysplasias and carcinomas compared with normal epithelial controls. Tumors with high Skp2 (>20% positive cells) expression invariably showed reduced or absent p27 and tumors with high p27 (>20% positive cells) expression rarely showed Skp2 positivity. Increased Skp2 protein levels were not always correlated with increased cell proliferation (assayed by Ki-67 staining), suggesting that alterations of Skp2 may contribute to the malignant phenotype without affecting proliferation. Skp2 protein overexpression may lead to accelerated p27 proteolysis and contribute to malignant progression from dysplasia to oral epithelial carcinoma. Moreover, we also demonstrate that Skp2 has oncogenic potential by showing that Skp2 cooperates with H-Ras G12V to malignantly transform primary rodent fibroblasts as scored by colony formation in soft agar and tumor formation in nude mice. The observations that Skp2 can mediate transformation and is up-regulated during oral epithelial carcinogenesis support a role for Skp2 as a protooncogene in human tumors.

A new cDNA encoding a protein of 362 amino acids designated SH3GLB1, for SH3 domain GRB2-like endophilin B1, was identified in a yeast two-hybrid screen devoted to the identification of new partners interacting with the apoptosis inducer Bax. SH3GLB1 shows strong similarities to the SH3 domain-containing proteins of the endophilin family and presumably represents the human homologue of the potential Caenorhabditis elegans SH3 containing-protein identified by systematic translation of the C. elegans genome (GenBank Accession No. U46675). Reversing prey to bait in the yeast screen, a second protein, SH3GLB2, of 395 amino acids showing 65% identity to SH3GLB1 was identified as an interacting partner of SH3GLB1. The discovery of SH3GLB1 itself in the screening with SH3GLB1 as a bait and further mapping experiments demonstrated that a core coiled-coil-type region is required for the formation of SH3GLB homo- and/or heterodimers, whereas the SH3 domain is not involved in these interactions. Interestingly, the similarities with the endophilin proteins cover the entire sequence of the SH3GLB family, suggesting a common fold and presumably a common mode of action. Furthermore, SH3GLB members colocalize to the cytoplasmic compartment of the cell together with Bax and are excluded from the nucleus. SH3GLB1 and SH3GLB2 do not significantly influence the onset and time course of Bax-mediated apoptosis in HeLa or 293T cells.

Successful treatment of chronic myelogenous leukemia is based on inhibitors binding to the ATP site of the deregulated breakpoint cluster region (Bcr)-Abelson tyrosine kinase (Abl) fusion protein. Recently, a new type of allosteric inhibitors targeting the Abl myristoyl pocket was shown in preclinical studies to overcome ATP-site inhibitor resistance arising in some patients. Using NMR and small-angle X-ray scattering, we have analyzed the solution conformations of apo Abelson tyrosine kinase (c-Abl) and c-Abl complexes with ATP-site and allosteric inhibitors. Binding of the ATP-site inhibitor imatinib leads to an unexpected open conformation of the multidomain SH3-SH2-kinase c-Abl core, whose relevance is confirmed by cellular assays on Bcr-Abl. The combination of imatinib with the allosteric inhibitor GNF-5 restores the closed, inactivated state. Our data provide detailed insights on the poorly understood combined effect of the two inhibitor types, which is able to overcome drug resistance.

CGP 41251 was originally identified as an inhibitor of protein kinase C (PKC), inhibiting mainly the conventional PKC subtypes, and subsequently shown to inhibit the vascular endothelial growth factor (VEGF) receptor kinase insert domain-containing receptor, which is involved in angiogenesis. CGP 41251 inhibits reversibly intracellular PKC activity, induction of c-fos and the corresponding activation of the mitogen-activated protein kinase induced by either tumor promoting phorbol esters, platelet-derived growth factor, or basic fibroblast growth factor, but not by the epidermal growth factor. CGP 41251 inhibited the ligand-induced autophosphorylation of the receptors for platelet-derived growth factor, stem cell factor, and VEGF (kinase insert domain-containing receptor) that correlated with the inhibition of the mitogen-activated protein kinase activation, but did not affect the ligand-induced autophosphorylation of the receptors for insulin, insulin-like growth factor-I, or epidermal growth factor. CGP 41251 showed broad antiproliferative activity against various tumor and normal cell lines in vitro, and is able to reverse the p-glycoprotein-mediated multidrug resistance of tumor cells in vitro. CGP 41251 showed in vivo antitumor activity as single agent and inhibited angiogenesis in vivo. Thus, CGP 41251 may suppress tumor growth by inhibiting tumor angiogenesis (via its effects on the VEGF receptor tyrosine kinases) in addition to directly inhibiting tumor cell proliferation (via its effects on PKCs).

On the basis of previously described X-ray studies of an enzyme/aza-dipeptide complex,8 aza-dipeptide analogues carrying N-(bis-aryl-methyl) substituents on the (hydroxethyl)hydrazine moiety have been designed and synthesized as HIV-1 protease inhibitors. By using either equally (12) or orthogonally (13) protected dipeptide isosteres, symmetrically and asymmetrically acylated aza-dipeptides can be synthesized. This approach led to the discovery of very potent inhibitors with antiviral activities (ED50) in the subnanomolar range. Acylation of the (hydroxethyl)hydrazine dipeptide isostere with the L-tert-leucine derivative 29 increased the oral bioavailability significantly when compared to the corresponding L-valine or L-isoleucine derivatives. The bis(L-tert-leucine) derivatives CGP 75355, CGP 73547, CGP 75136, and CGP 75176 combine excellent antiviral activity with high blood concentration after oral administration. Furthermore, they show no cross-resistance with saquinavir-resistant strains and maintain activity against indinavir-resistant ones. Consequently they qualify for further profiling as potential clinical candidates.

Protein binding can impair the potency of human immunodeficiency virus (HIV) protease inhibitors. Therefore, the activity of a novel compound, CGP 61755, was studied in the absence or presence of alpha1-acid glycoprotein (alpha1AGP). In MT-2 cells, the activity loss was 4-fold (EC90 without alpha1AGP, 29 nM vs. 122 nM with alpha1AGP). In primary lymphocytes, the loss was 8-fold (EC90, 45 nM vs. 364 nM). In identical experiments, the activity loss in MT-2 cells and lymphocytes was 2- and 3-fold, respectively, for indinavir, 11- and 10-fold for saquinavir, and 11- and 48-fold for ritonavir. For SC-52151, a 17-fold loss was seen in MT-2 cells, whereas no EC90 with alpha1AGP was reached in lymphocytes. This study demonstrates that the impact of alpha1AGP on in vitro activity varies greatly among different HIV protease inhibitors. The magnitude of such differences is greater in human lymphocytes than in a standard cell line.

A series of aza-peptide analogs with a (hydroxyethyl)hydrazine isostere has been synthesized as HIV-1 protease inhibitors using a simple synthetic scheme. Structure-activity studies based on the X-ray of a previously described inhibitor-enzyme complex led to potent inhibitors with antiviral activity in the low-nanomolar range. The S-configuration of the transition-state hydroxyl group was preferred in this series. Small modifications of the P2P3 and P2'P3' substituents had little effect on enzyme inhibition but greatly influenced the pharmacokinetic profile. As a result of these studies, the symmetrically acylated compound 8a and its close analog 24a bearing a methyl carbamate in P3 and an ethyl carbamate in P3' position were identified as potent inhibitors with plasma concentrations exceeding antiviral ED50 values 150-fold following oral application in mice.

A series of potent HIV-protease inhibitors has been prepared. Several of the newly synthesized compounds showed high plasma levels after oral administration to animals. Based on the overall biological profile, CGP 61755 was chosen for further preclinical evaluation. For this compound, a 10 step synthesis potentially suitable for large scale production was developed.

Mammalian target of rapamycin (mTOR) signaling pathway plays a fundamental role in cell proliferation, differentiation, growth and survival.[1] As a consequence, various tumors and central nervous system (CNS) disorders share aberrant activation of the mTOR pathway. Drugs targeting the mTOR pathway represent therefore a valuable path to address multiple therapeutic areas.[1-2] Here, we report the lead optimization of PQR620, a novel potent and selective brain penetrant inhibitor of mTORC1/2. The development of selective mTOR inhibitors is particularly challenging due to extensively conserved amino acid residues in the ATP binding pocket within the PI3K and PI3K-related protein kinase family. Here, we present a detailed ligand-based structure activity relationship study allowing selective targeting of mTOR kinase activity without the interference of other PI3K family members. Systematic variation of the hinge region and affinity binding motifs led to the identification of PQR620, a morpholino-triazinyl derivative, as potent and selective mTOR inhibitor. Substitution of the morpholine binding to the hinge region and introduction of a 2-aminopyridine, substituted with a difluoromethyl group, induced a >1000-fold selectivity towards mTOR over PI3Kα in enzymatic binding assays. In A2058 melanoma cells PQR620 demonstrated inhibition of protein kinase B (pSer473) and ribosomal protein S6 (pSer235/236) phosphorylation with IC50 values of 0.2 μM and 0.1 μM, respectively. The physico-chemical properties of PQR620 result in good oral bioavailability and excellent brain penetration. PQR620 showed excellent selectivity over a wide panel of kinases, as well as excellent selectivity versus unrelated receptor enzymes and ion channels. Moreover, PQR620 demonstrated its potency to prevent cancer cell growth in an NTRC 44 cancer cell line panel, resulting in a 10log(IC50) of 2.86 (nM). Further pharmacological properties and in vivo efficacy of PQR620 are presented in detail in Ref. [3]. The preparation of PQR620 was optimized towards a robust synthetic route involving only 4 steps, allowing for a rapid access to quantities required for pre-clinical testing. In conclusion, PQR620 inhibits mTOR potently and selectively, and shows anti-tumor effects in vitro and in vivo. PQR620 is currently in pre-clinical development. [1] M. Laplante, D. Sabatini, Cell 2012, 149, 274-293. [2] Z. Z. Chong, Y. C. Shang, L. Zhang, S. Wang, K. Maiese, Oxid. Med. Cell. Longev. 2010, 3, 374–391. [3] F. Beaufils, D. Rageot, A. Melone, A. M. Sele, M. Lang, J. Mestan, R. A. Ettlin, P. Hillmann, V. Cmiljanovic, C. Walter, E. Singer, H. P. Nguyen, P. Hebeisen, D. Fabbro, M. P. Wymann, “Pharmacological characterization of the selective, orally bioavailable, potent mTORC1/2 inhibitor PQR620” presented at AACR Annual Meeting 2016, April 16-20, New Orleans, Louisiana, USA. Citation Format: Florent Beaufils, Denise Rageot, Anna Melone, Marc Lang, Jürgen Mestan, Vladimir Cmiljanovic, Petra Hillmann, Paul Hebeisen, Doriano Fabbro, Matthias P. Wymann. Structure-activity relationship studies, synthesis, and biological evaluation of PQR620, a highly potent and selective mTORC1/2 inhibitor. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1336.

The second generation of Bcr-Abl inhibitors nilotinib, dasatinib, and bosutinib developed to override imatinib resistance are not active against the T315I “gatekeeper” mutation. Here we describe a type-II T315I inhibitor 2 (GNF-7), based upon a 3,4-dihydropyrimido[4,5-d]pyrimidin-2(1H)-one scaffold which is capable of potently inhibiting wild-type and T315I Bcr-Abl as well as other clinically relevant Bcr-Abl mutants such as G250E, Q252H, Y253H, E255K, E255V, F317L, and M351T in biochemical and cellular assays. In addition, compound 2 displayed significant in vivo efficacy against T315I-Bcr-Abl without appreciable toxicity in a bioluminescent xenograft mouse model using a transformed T315I-Bcr-Abl-Ba/F3 cell line that has a stable luciferase expression. Compound 2 is among the first type-II inhibitors capable of inhibiting T315I to be described and will serve as a valuable lead to design the third generation Bcr-Abl kinase inhibitors.

As a drug used to treat imatinib-resistant and -intolerant, chronic and advanced phase chronic myelogenous leukaemia, nilotinib is well characterised as a potent inhibitor of the Abl tyrosine kinase activity of wild-type and imatinib-resistant mutant forms of BCR-Abl. Here we review the profile of nilotinib as a protein kinase inhibitor. Although an ATP-competitive inhibitor of Abl, nilotinib binds to a catalytically inactive conformation (DFG-out) of the activation loop. As a consequence of this, nilotinib exhibits time-dependent inhibition of Abl kinase in enzymatic assays, which can be extrapolated to other targets to explain differences between biochemical activity and cellular assays. Although these differences confound assessment of kinase selectivity, as assessed using a combination of protein binding and transphosphorylation assays, together with cellular autophosporylation and proliferation assays, well established kinase targets of nilotinib in rank order of inhibitory potency are DDR-1 > DDR-2 > BCR-Abl (Abl) > PDGFRα/β > KIT > CSF-1R. In addition nilotinib has now been found to bind to both MAPK11 (p38β) and MAPK12 (p38α), as well as with very high affinity to ZAK kinase. Although neither enzymatic nor cellular data are yet available to substantiate the drug as an inhibitor of ZAK phosphorylation, modeling predicts that it binds in an ATP-competitive fashion.

The discovery of STI571, a drug targeting the tyrosine kinase activity of Bcr-Abl, c-Kit, and platelet-derived growth factor receptor (PDGFR), has demonstrated the feasibility of ATP-competitive small-molecule kinase inhibitors for the chronic treatment of molecularly defined cancers. The presence of an activated form of protein kinases targeted by STI571 in various malignancies appears to be mandatory for a clinical response to this drug. This also indicates that a certain subset of molecularly defined tumors depends upon the overactivation of one or more signaling pathways, which can be attacked by targeted therapy. The finding that STI571 resistance is often associated with mutations in the kinase domain unambiguously demonstrates that the targets of STI571 in these tumors are Bcr-Abl, c-Kit, and/or PDGFR. Generation of resistance to STI571 is most likely due to the binding mode of STI571 that stabilizes the inactive conformation of the target kinases. Recent mutational analysis combined with structural biology has provided the basis for the understanding of the binding mode of STI571, and the resistance mechanisms as well as activation mechanisms of Bcr-Abl, c-Kit, and PDGFR. Although this binding mode is key for the exquisite selectivity and tolerability of STI571, it is also its “weakness”, because it allows mutations to occur in the target kinase which appear not to affect the normal function of the kinase. Therefore, for an effective long-term cancer therapy with ATP-competitive small-molecule kinase inhibitors, it appears necessary to use more than one kinase inhibitor targeting different conformations of the same kinase, or each inhibitor needs to be aimed at a specific mutant and downstream signaling element.

Resistance to or intolerance of imatinib in patients with Philadelphia chromosome–positive chronic myelogenous leukemia (CML) has encouraged the development of more potent Bcr-Abl inhibitors. AMN107 is a novel, orally bioavailable ATP-competitive inhibitor of Bcr-Abl. The effects of AMN107 were compared with those of imatinib on imatinib-sensitive (KBM5 and KBM7) and imatinib-resistant CML cell lines (KBM5-STI571R1.0 and KBM7-STI571R1.0). Compared with the antiproliferative activity of imatinib, AMN107 was 43 times more potent in KBM5 (IC50 of 11.3 versus 480.5 nmol/L) and 60 times more potent in KBM7 (IC50 of 4.3 versus 259.0 nmol/L) cells. IC50 for AMN107 and imatinib were 2,418.3 and 6,361.4 nmol/L, respectively, in KBM5-STI571R1.0, and 97.2 and 2,497.3 nmol/L, respectively, in KBM7-STI571R1.0 cells. AMN107 inhibited autophosphorylation of Bcr-Abl kinase more effectively than imatinib in all cell lines. They had similar effects on cell cycle progression and apoptotic response in these cell lines. Among severe combined immunodeficient mice bearing KBM5 cells, mean survival times of groups treated with 10, 20, and 30 mg/kg/d of AMN107, starting day 20 after leukemic cell grafting and continuing for 20 days, were 144%, 159%, and 182%, respectively, compared with controls. These results strongly support investigation of the clinical efficacy of AMN107 in patients with CML.

The constitutively activated Abl tyrosine kinase domain of the chimeric Bcr-Abl oncoprotein is responsible for the transformation of haematopoietic stem cells and the symptoms of chronic myeloid leukaemia (CML). Imatinib targets the tyrosine kinase activity of Bcr-Abl and is a first-line therapy for this malignancy. Although highly effective in chronic phase CML, patients who have progressed to the advanced phase of the disease frequently fail to respond to imatinib or develop resistance to therapy and relapse. This is often due to the emergence of clones expressing mutant forms of Bcr-Abl, which exhibit a decreased sensitivity towards inhibition by imatinib. Considerable progress has recently been made in understanding the structural biology of Abl and the molecular basis for resistance, facilitating the discovery and development of second generation drugs designed to combat mutant forms of Bcr-Abl. The first of these compounds to enter clinical development were BMS-354825 (BristolMyersSquibb) and AMN107 (Novartis Pharma) and, from Phase I results, both of these promise a breakthrough in the treatment of imatinib-resistant CML. Recent advances with these and other promising classes of new CML drugs are reviewed.

SummaryThe Bcr-Abl tyrosine kinase oncogene causes chronic myelogenous leukemia (CML) and Philadelphia chromosome-positive (Ph+) acute lymphoblastic leukemia (ALL). We describe a novel selective inhibitor of Bcr-Abl, AMN107 (IC50 < 30 nM), which is significantly more potent than imatinib, and active against a number of imatinib-resistant Bcr-Abl mutants. Crystallographic analysis of Abl-AMN107 complexes provides a structural explanation for the differential activity of AMN107 and imatinib against imatinib-resistant Bcr-Abl. Consistent with its in vitro and pharmacokinetic profile, AMN107 prolonged survival of mice injected with Bcr-Abl-transformed hematopoietic cell lines or primary marrow cells, and prolonged survival in imatinib-resistant CML mouse models. AMN107 is a promising new inhibitor for the therapy of CML and Ph+ ALL.

Aberrant epidermal growth factor receptor (EGFR) and ErbB2 expression are associated with advanced disease and poor patient prognosis in many tumor types (breast, lung, ovarian, prostate, glioma, gastric, and squamous carcinoma of head and neck). In addition, a constitutively active EGFR type III deletion mutant has been identified in non-small cell lung cancer, glioblastomas, and breast tumors. Hence, members of the EGFR family are viewed as promising therapeutic targets in the fight against cancer. In a similar vein, vascular endothelial growth factor (VEGF) receptor kinases are also promising targets in terms of an antiangiogenic treatment strategy. AEE788, obtained by optimization of the 7H-pyrrolo[2,3-d]pyrimidine lead scaffold, is a potent combined inhibitor of both epidermal growth factor (EGF) and VEGF receptor tyrosine kinase family members on the isolated enzyme level and in cellular systems. At the enzyme level, AEE788 inhibited EGFR and VEGF receptor tyrosine kinases in the nm range (IC(50)s: EGFR 2 nm, ErbB2 6 nm, KDR 77 nm, and Flt-1 59 nm). In cells, growth factor-induced EGFR and ErbB2 phosphorylation was also efficiently inhibited (IC(50)s: 11 and 220 nm, respectively). AEE788 demonstrated antiproliferative activity against a range of EGFR and ErbB2-overexpressing cell lines (including EGFRvIII-dependent lines) and inhibited the proliferation of epidermal growth factor- and VEGF-stimulated human umbilical vein endothelial cells. These properties, combined with a favorable pharmacokinetic profile, were associated with a potent antitumor activity in a number of animal models of cancer, including tumors that overexpress EGFR and or ErbB2. Oral administration of AEE788 to tumor-bearing mice resulted in high and persistent compound levels in tumor tissue. Moreover, AEE788 efficiently inhibited growth factor-induced EGFR and ErbB2 phosphorylation in tumors for72 h, a phenomenon correlating with the antitumor efficacy of intermittent treatment schedules. Strikingly, AEE788 also inhibited VEGF-induced angiogenesis in a murine implant model. Antiangiogenic activity was also apparent by measurement of tumor vascular permeability and interstitial leakage space using dynamic contrast enhanced magnetic resonance imaging methodology. Taken together, these data indicate that AEE788 has potential as an anticancer agent targeting deregulated tumor cell proliferation as well as angiogenic parameters. Consequently, AEE788 is currently in Phase I clinical trials in oncology.

Initial studies with angiogenesis inhibitors showed little clinical benefit. However, recently reported clinical studies in colorectal cancer have shown that bevacizumab, a vascular endothelial growth factor (VEGF) monoclonal antibody, in combination with cytotoxic therapy has positive effects on patient survival. Furthermore, the VEGF receptor kinase (VEGF-R) tyrosine kinase inhibitor, vatalanib, has also shown encouraging results in colorectal cancer, with molecular resonance imaging providing evidence that the anti-tumor efficacy was indeed the result of anti-angiogenic activity. Both of these agents are progressing in phase III trials. This proof of concept has stimulated the desire for second-generation VEGF-R inhibitors having an improved profile. Structural biology insight regarding the binding mode of protein kinase inhibitors is valuable for the design of molecules possessing superior selectivity, efficacy and tolerability. Towards this goal, we have developed a new series of VEGF-R2 kinase inhibitors, based upon an anthranilic acid amide scaffold. An X-ray crystal structure of a representative compound, AAL993 (ZK260253), in complex with the catalytic domain of diphosphorylated VEGF-R2 has revealed that this molecule binds to an inactive conformation of the protein. This binding mode, similar to that observed for the anti-leukemia drug, imatinib in complex with c-Abl kinase, may be responsible for the high selectivity of AAL993 and provides valuable insight for the design of further compounds.

Two readily synthesized anthranilamide, VEGF receptor tyrosine kinase inhibitors have been prepared and evaluated as angiogenesis inhibitors. 2-[(4-Pyridyl)methyl]amino-N-[3-(trifluoromethyl)phenyl]benzamide (5) and N-3-isoquinolinyl-2-[(4-pyridinylmethyl)amino]benzamide (7) potently and selectively inhibit recombinant VEGFR-2 and VEGFR-3 kinases. As a consequence of their physicochemical properties, these anthranilamides readily penetrate cells and are absorbed following once daily oral administration to mice. Both 5 and 7 potently inhibit VEGF-induced angiogenesis in an implant model, with ED(50) values of 7 mg/kg. In a mouse orthotopic model of melanoma, 5 and 7 potently inhibited both the growth of the primary tumor as well as the formation of spontaneous peripheral metastases. The anthranilamides 5 and 7 represent a new structural class of VEGFR kinase inhibitors, which possess potent antiangiogenic and antitumor properties.

Many components of mitogenic signaling pathways in normal and neoplastic cells have been identified, including the large family of protein kinases, which function as components of signal transduction pathways, playing a central role in diverse biological processes, such as control of cell growth, metabolism, differentiation, and apoptosis. The development of selective protein kinase inhibitors that can block or modulate diseases caused by abnormalities in these signaling pathways is widely considered a promising approach for drug development. Because of their deregulation in human cancers, protein kinases, such as Bcr-Abl, those in the epidermal growth factor-receptor (HER) family, the cell cycle regulating kinases such as the cyclin-dependent kinases, as well as the vascular endothelial growth factor-receptor kinases involved in the neo-vascularization of tumors, are among the protein kinases considered as prime targets for the development of selective inhibitors. These drug-discovery efforts have generated inhibitors and low-molecular weight therapeutics directed against the ATP-binding site of various protein kinases that are in various stages of development (up to Phase II/III clinical trials). Three examples of inhibitors of protein kinases are reviewed, including low-molecular weight compounds targeting the cell cycle kinases; a potent and selective inhibitor of the HER1/HER2 receptor tyrosine kinase, the pyrollopyrimidine PKI166; and the 2-phenyl-aminopyrimidine STI571 (Glivec(R), Gleevec) a targeted drug therapy directed toward Bcr-Abl, the key player in chronic leukemia (CML). Some members of the HER family of receptor tyrosine kinases, in particular HER1 and HER2, have been found to be overexpressed in a variety of human tumors, suggesting that inhibition of HER signaling would be a viable antiproliferative strategy. The pyrrolo-pyrimidine PKI166 was developed as an HER1/HER2 inhibitor with potent in vitro antiproliferative and in vivo antitumor activity. Based upon its clear association with disease, the Bcr-Abl tyrosine kinase in CML represents the ideal target to validate the clinical utility of protein kinase inhibitors as therapeutic agents. In a preclinical model, STI571 (Glivec(R), Gleevec) showed potent in vitro and in vivo antitumor activity that was selective for Abl, c-Kit, and the platelet-derived growth factor-receptor. Phase I/II studies demonstrated that STI571 is well tolerated, and that it showed promising hematological and cytogenetic responses in CML and clinical responses in the c-Kit-driven gastrointestinal tumors.

Background There is a growing tendency for drugs to be grouped according to their perceived ‘class effects’, regardless of the different pharmacological profiles of the parent drugs and of their metabolites. Imatinib, dasatinib, nilotinib, bosutinib and, most recently ponatinib, are approved tyrosine kinase inhibitor (TKI) therapies for chronic myeloid leukemia (CML), which are clinically efficacious as a result of ABL1/ BCR-ABL inhibition. Following their oral administration at standard therapeutic doses, the parent drugs are the major circulating species by area under the curve (AUC). However in the case of imatinib, dasatinib, bosutinib and ponatinib, the exposure of patients to major metabolites can be substantial compared to that of parent drug, with CGP74588 (which is much less active than imatinib against both BCR-ABL and KIT; Bioorg Med Chem 2013;21:3231) representing 10% of imatinib by AUC (Clin Pharmacokinet 2005;44:879); M20 and M24 representing 45 and 25% of dasatinib (Drug Met Disp 2008;36:1341), M2 and M5 representing 19 and 25% of bosutinib (Clinical Pharmacology Biopharmaceutics Review, http://www.accessdata.fda.gov/scripts/cder/drugsatfda/index.cfm) and AP24600 representing 58% of ponatinib (Clinical Pharmacology Biopharmaceutics Review, http://www.accessdata.fda.gov/scripts/cder/drugsatfda/index.cfm). Such major metabolites might make significant contributions to the on- and off-target effects of the parent drugs in vivoand may be responsible for some of the side-effects observed in patients. Here we report on the metabolism of the potent and selective BCR-ABL inhibitor, nilotinib and the preclinical profile of its major metabolites. Methods The metabolism of nilotinib was characterised in healthy subjects after oral administration of two capsules containing 200 mg [14C]-labelled nilotinib (50 μCi), and blood plasma, feces and urine samples were assayed in an appropriate scintillant either by counting an aliquot directly or after homogenisation, air-drying and solubilisation. Metabolites were characterised and quantified by HPLC with radioactivity detection and identified by mass spectrometry (LC-MS/MS) and, when possible, co-elution with non-radiolabeled authentic samples. Synthesised samples of the metabolites were evaluated in a large panel of assays for potential effects on kinase and non-kinase enzymes, G-protein coupled receptors, cell transporters, ion channels and nuclear receptors. Results The oral absorption of nilotinib was determined to be ≥30% and excretion was mainly into the feces (93.5% of administered radioactivity), with neither nilotinib nor the identified metabolites being detected in the urine. Unchanged nilotinib was the major circulating component in human plasma, accounting for 87.5±9.2% of the total drug-related AUC. The main circulating metabolites were P41.6 (4.7% AUC), P36.5 (6.1% AUC), formed from oxidation of the methyl group in the methyl-imidazole moiety to a hydroxyl or carboxylic acid group, and P42.1 (1.3% AUC) resulting from oxidation of the phenyl-methyl group. Other, more minor metabolites included the pyridine N-oxide P36 and P50, resulting from degradation of the imidazole. All of the metabolites identified in humans were also observed in one or more of the animal species, employed for preclinical safety studies, with the exception of the minor fecal metabolites P38 (pyridine- + pyrimidine-N-oxide) and P40 (pyridine-N-oxide). In comparison to the parent nilotinib, which inhibits the BCR-ABL and KIT tyrosine kinases with mean cellular IC50 values of 20 and 217 nM, only P41.6 (19 and 284 nM), P42.1 (256 and 714 nM) and P50 (39 and 67 nM) exhibited kinase inhibition at concentrations < 2200 nM. In addition, none of the metabolites showed substantial activity at concentrations < 3000 nM against non-kinase targets. Conclusion Following oral administration of nilotinib to humans the predominant circulating species was the parent drug, with >15 minor and trace metabolites being identified. Given their in vitro potencies and target profiles, none of the metabolites are expected to contribute to the in vivo pharmacology of the parent nilotinib. This data further distinguishes the profile of nilotinib from other TKIs used for the treatment of CML. Disclosures: Manley: Novartis Pharmaceuticals: Employment. Sheng:Novartis Pharmaceuticals: Employment. Tran:Novartis Pharmaceuticals: Employment. Kagan:Novartis Pharmaceuticals: Employment.

The sprouting of new blood vessels, or angiogenesis, is necessary for any solid tumor to grow large enough to cause life-threatening disease. Vascular endothelial growth factor (VEGF) is one of the key promoters of tumor induced angiogenesis. VEGF receptors, the tyrosine kinases Flt-1 and KDR, are expressed on vascular endothelial cells and initiate angiogenesis upon activation by VEGF. 1-Anilino-(4-pyridylmethyl)-phthalazines, such as CGP 79787D (or PTK787 / ZK222584), reversibly inhibit Flt-1 and KDR with IC(50) values0.1 microM. CGP 79787D also blocks the VEGF-induced receptor autophosphorylation in CHO cells ectopically expressing the KDR receptor (ED(50) = 34 nM). Modification of the 1-anilino moiety afforded derivatives with higher selectivity for the VEGF receptor tyrosine kinases Flt-1 and KDR compared to the related receptor tyrosine kinases PDGF-R and c-Kit. Since these 1-anilino-(4-pyridylmethyl)phthalazines are orally well absorbed, these compounds qualify for further profiling and as candidates for clinical evaluation.

Polyclonal antibodies to native chicken pectoral fast-twitch myosin are directed to all subfragments of the molecule (S1, S2 and LMM), as seen in the ELISA and Western blotting techniques. The antibodies inhibit the Ca(2+)-activated myosin ATPase. Absorption of the antibodies with native myosin abolishes these reactions. Heat treatment of myosin for 2h at 40 degrees C will inactivate myosin ATPase and alter its antibody binding pattern: the binding of antibodies to the rod fractions is reduced, that to the globular head (S1) completely abolished. Thus, these antibodies are useful as sensitive probes for the structural integrity of the myosin head.

Abstract 510 Background: Recently, Redaelli et al (J Clin Oncol. 2009;27:469) compared the in vitro inhibitory activity of imatinib, dasatinib, nilotinib, and bosutinib against 18 mutant forms of BCR-ABL (expressed in transfected Ba/F3 cells) associated with imatinib resistance and proposed a chart to assist in the selection of second-generation tyrosine kinase inhibitors (2TKIs) for the treatment of imatinib-resistant CML associated with mutations. However, the predictability of this chart has neither been clinically evaluated nor does it take into account other important clinical factors (e.g. pharmacokinetics (PK)/pharmacodynamics) that may impact response rates to 2TKIs in the presence of mutations. The purpose was to assess the impact of 2TKIs' in vivo plasma levels on the in vitro GI50 data, and to determine if in vitro GI50 data with or without plasma levels correlates with observed clinical responses in imatinib-resistant patients (pts) with mutations. Methods: To enable appropriate comparison of the activity of 2TKIs against specific mutations we modified the original in vitro GI50 data by adjusting it to include an estimate of in vivo Cmax exposure data for each 2TKI. Further refinement was achieved by calculating the Cmax/GI50 values for each agent and normalizing these against imatinib vs wild-type BCR-ABL. To assess the correlation between patient response and in vitro GI50 data, the previously published CCyR rates for pts with specific mutations were plotted according to in vitro GI50 values alone and against the adjusted Cmax/GI50 values. Results: The adjusted Cmax/GI50 data suggest that nilotinib delivers the most potent inhibition of most BCR-ABL mutations in vivo, with dasatinib being the next most potent. However, when either in vitro GI50 data alone or the modified Cmax/GI50 data are considered, there is poor correlation of clinical responses to both nilotinib and dasatinib against several of the mutations in vivo (Figure). Overall, activity of 2TKIs against all mutations was less than expected based on original in vitro GI50 or Cmax/GI50 calculations of systemic exposure. For example, the G250E mutation has similar systemic exposure to nilotinib as the F359V mutation as indicated by Cmax/GI50, but substantial differences are observed in the CCyR rate (60% vs 0%). For dasatinib, the same was observed for the F317L and L248V mutations which have similar exposures to dasatinib but have different CCyR rates (7% vs 41%). Similarly, several mutations with comparable exposure to nilotinib and dasatinib had substantial differences in CCyR rates, suggesting that other factors were influencing responses. For example, the G250E mutation was considered moderately sensitive to both nilotinib and dasatinib based on the adjusted Cmax/GI50; however, CCyR rates on nilotinib were much higher (60%) compared with dasatinib (34%). Similarly, the E255K mutation was considered moderately sensitive to both agents based on the adjusted Cmax/GI50; however, CCyR rates on dasatinib were much higher (38%) compared with nilotinib (13%). Conclusions: This analysis illustrates the limitations of in vitro inhibition data alone or in combination with PK exposure data in the selection of 2TKI therapy for imatinib-resistant pts with mutations. The current analysis still does not consider parameters such as protein binding and intracellular influx/efflux, among a variety of other clinical factors that could further influence response rates. This tool is also not useful for pts with mutations of unknown in vitro sensitivity, which may represent 15% of all pts with mutations. Currently, clinical responses remain the best approach for selection of 2TKIs in pts with mutations, with only a small subset of mutations having low sensitivity mutations existing for each 2TKI. Other factors, such as patient medical history, comorbidities, and the agents' safety profiles, are also important in selection of 2TKIs. Disclosures: Laneuville: Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Bristol-Myers Squibb: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Wyeth: Research Funding; ChemGenex: Research Funding. DiLea:Novartis: Employment. Mestan:Novartis: Employment. Yin:Novartis: Employment, Equity Ownership. Woodman:Novartis: Employment. Manley:Novartis: Employment.

AMN107 (Novartis Pharma AG), is a new, highly potent and selective inhibitor of the Abl tyrosine kinase activity of the Bcr-Abl oncoprotein, which causes chronic myelogenous leukemia (CML). In addition to inhibiting wild-type Bcr-Abl, AMN107 inhibits the activity of 32/33 mutant forms of this protein which occur in imatinib-resistant patients. Following promising results from Phase I clinical studies in imatinib-resistant CML patients, AMN107 has now entered Phase II clinical trials. In order to better understand the molecular basis for the activity of this compound we have examined the x-ray crystallographic structures of complexes between AMN107 and the Abl kinase domain of wild-type and mutant Bcr-Abl. For these studies, recombinant proteins (residues 229–500) corresponding to the wild type and M351T mutant of the human Abl kinase domain were expressed in baculovirus infected insect cells and purified in the presence of AMN107. In each case the crystals obtained contained four independent copies of the complex in the unit cell and they diffracted to a resolution of 2.2 and 2.7 Å, respectively. As has been reported for the prototype Bcr-Abl inhibitor, imatinib (Nagar et al., Cancer Research2002;62:4236), AMN107 binds to an inactive conformation of Abl, in which the glycine-rich, P-loop folds over the ATP binding site and the activation-loop adopts a conformation in which it occludes the substrate binding site and disrupts the ATP-phosphate binding site to block the catalytic activity of the enzyme. In order to induce the inactive conformation of the protein, AMN107 participates in hydrogen-bond interactions between (i) the pyridine-N and the backbone-C=O of Met318, (ii) the anilino-NH and the side-chain hydroxyl of Thr315, (iii) the carboxamido-C=O and the backbone-NH of Asp381, and (iv) the carboxamido-NH and the side-chain carboxylate of Glu286. These hydrogen-bond interactions are complemented by a large number of lipophilic interactions, surrounding the pyridine and 4-methylimidazole moieties in particular. Furthermore, careful analysis has revealed the presence of interactions between the protein and the trifluoromethyl group of AMN107, in which a fluorine atom is in close contact with the backbone-C=O of Asp381, with a mean F-C distance of 3.02 Å (4 mols/unit cell: 2.95, 2.97, 3.08 and 3.09). This compares with a value of 3.30 Å for the sum of the van der Waals radii of fluorine and carbon. Similar F-C=O interactions (mean F-C distance 3.02 Å) are observed in a complex between AMN107 and M351TAbl. Such interactions have been reported in the literature (Olsen et al., ChemBioChem2004;5:666) and are the result of dipolar interactions between the electronegative fluorine and the positively polarised carbon of the carbonyl group. In order to evaluate the contribution of the fluorine interactions to the binding of AMN107 to Abl we compared the effects of AMN107 on Bcr-Abl autophosphorylation in Ba/F3 cells to those of the corresponding analogue of AMN107, possessing a methyl group in place of trifluoromethyl. Whereas AMN107 displayed a mean IC50 value of 17 ± 0.5 nM, the methyl analogue had a mean IC50 of 83 ± 13 nM, which translates into a difference in binding energy in the region of 0.7 kcal/mole. Although Asp381 is therefore important for the binding of AMN107 to Abl through a hydrogen bond and through F-C interactions, resistance-mutations of this residue are unlikely the arise, since it plays a key role in the catalytic activity of the enzyme by interacting with a Mg ion which coordinates the phosphate groups of ATP.

6522 Background: The Bcr-Abl oncoprotein has unregulated tyrosine kinase activity and causes chronic myelogenous leukemia (CML). Imatinib, a Bcr-Abl kinase inhibitor, is an effective therapy. Howev...

The selective tyrosine kinase inhibitor imatinib eradicates bcr-abl+ cells in chronic myeloid leukemia patients (pts). Although a previous clinical trial showed superiority of an imatinib therapy over an interferon-α containing regimen, a significant number of pts eventually relapse with leukemia because of either point mutations within the imatinib-binding site, amplification of the Philadelphia chromosome or other mechanisms, e.g. clonal evolution. AMN107 (Novartis Pharma AG) is a new anilino-pyrimidine derivative (MW: 529.5) structurally related to imatinib. AMN107 was tested in three human bcr-abl positive lines (K562, KCL-22, Lama-84) and in primary cells derived from two bcr-abl + CML pts who were resistant to imatinib, as well as in one newly diagnosed chronic phase patient. In all pts sequencing of the bcr-abl kinase domain excluded any point mutations, but cytogenetic analysis of the bone marrow revealed clonal evolution in the resistant pts including t(1;5) and t(3;21) translocations, trisomy of chromosome 8 and monosomy of chromosome 7. Determination of the proliferative activity by XTT-assay in cell lines demonstrated a decrease of the IC50 in imatinib versus AMN107 treated samples from 0.08μM to 0.0075μM in Lama 84, from 0.25μM to 0.08μM in K562 and from 0.45μM to 0.03 in KCL-22 cells. No activity of either compound was observed in the bcr-abl negative HL-60 and KG-1 cells. In primary cells from imatinib-resistant pts, a decrease of the IC50 in imatinib versus AMN107 treated peripheral blood cells from 0.75μM to 0.1μM and from 4 to 0.4μM was detected. In addition, in primary cells from one newly diagnozed CML patient the IC50 of AMN107 (2.5μM) was reduced when compared to imatinib (5μM). Immunoblotting showed that in LAMA84 cells a concentration of 0.01μM AMN107 completely inhibited the tyrosine kinase activity as detected by use of an anti-phosphotyrosine antibody in contrast to almost 5μM in imatinib treated samples. Further, induction of apoptosis was detected using annexin V and propidium iodide by double fluorescence. After 48 hours of incubation with either 0.25 μM imatinib or 0.005 μM AMN107 induction of early apoptosis was detected in 8.8% of imatinib treated and 26% of AMN107 treated cells. Finally, HPLC analysis in HL-60 cells showed increased uptake by 1,5 fold for AMN107 when compared to imatinib. In addition, in MDR1 over-expressing CCRF cells co-culture with either AMN107 or imatinib revealed elevated AMN107 levels (3.7 fold) indicating that this substance is less susceptible to MDR1 driven resistance than imatinib. Conclusions: 1. AMN107 showed elevated activity when compared to imatinib in bcr-abl + cell lines and primary cells derived from imatinib resistant leukemic pts. 2. Complete inhibition of the bcr-abl tyrosine kinase activity and induction of apoptosis was achieved at lower concentrations in AMN107 treated samples when compared to imatinib. 3. Preliminary data indicate favourable cellular uptake of AMN107 when compared to imatinib. 4. AMN107 may be useful in the treatment of bcr-abl + leukemic pts.

Patient relapse due to the emergence of imatinib-resistance in advanced chronic myelogenous and Ph+ acute lymphocytic leukemias has prompted the search for improved Bcr-Abl kinase inhibitors. Up to 40% imatinib-resistant patients express mutant forms of the Bcr-Abl kinase that reduce binding of imatinib without significantly decreasing kinase activity. However, some mutants are only partially resistant, since higher concentrations of imatinib may still block kinase activity and have a beneficial clinical effect. More potent Bcr-Abl inhibitors or inhibitors with different contact points with the kinase domain are predicted to be useful for the treatment of imatinib-resistant disease. Rational drug design based upon the crystal structure of an Abl-imatinib complex, together with medicinal chemistry paradigms, resulted in the discovery of AMN107. AMN107 is a low molecular weight compound that selectively inhibits the c-Abl/Bcr-Abl, imatinib-resistant mutants of Bcr-Abl, PDGFR, and c-Kit tyrosine kinases, with IC50 values for the inibition of autophosphorylation (cell capture ELISA) of 23 nM (32D cells transfected with p210 Bcr-Abl), 83 nM (A31 cells) and 192 nM (GIST cells), respectively. Following oral administration to mice (20 mg/kg in NMP-PEG300 10:90 v/v), AMN107 was well absorbed, with a mean plasma level after 2 h in the range of 6.0-12.1 M, which corresponds to >100-fold that required to inhibit Bcr-Abl autophosphorylation in 32D.p210 cells. Following injection of 32D.p210-luciferase cells into Balb/c mice, serial imaging of the leukemic clone in live mice indicated that AMN107 could substantially reduce the accumulation of leukemic cells in the marrow, nodes, liver, and spleen, compared to a vehicle control. Further, administration of AMN107 (75 mg/kg/day p.o.) to BALB/c mice over an 18 day period, commencing 3 days after the injection of 32 D.p210 cells, resulted in the protection of 15/20 animals over 100 days observation, whereas 19/20 vehicle treated animals developed a lethal leukemic disease(all deaths occurred within 36 days of cell injection). The spleen sizes of the surviving animals in the treated group were in the normal range (0.142±0.043 g; n = 17), whereas those of the vehicle treated animals were 0.50±0.045 (n = 20). These results were confirmed in a bone marrow transplant assay, where AMN107 was found to prolong survival of mice transplanted with marrow cells infected with a p210Bcr/Abl retrovirus. AMN107 also prolonged survival of mice transplanted with a Bcr/Abl mutant (E255V) associated with imatinib resistance in patients, while imatinib treatment was unsuccessful. These in vivo studies, along with in vitro studies reported separately, indicate that AMN107 is a highly active inhibitor of Bcr/Abl that may have clinical utility in patients with Bcr/Abl+ leukemias.