Search

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.

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

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.

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.

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).

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.

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.

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.

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.

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.

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.