Frank Boschelli

In vitro and in vivo activity of SKI-606, a novel Src-Abl inhibitor, against imatinib-resistant Bcr-Abl + neoplastic cells

Resistance to imatinib represents an important scientific and clinical issue in chronic myelogenous leukemia. In the present study, the effects of the novel inhibitor SKI-606 on various models of resistance to imatinib were studied. SKI-606 proved to be an active inhibitor of Bcr-Abl in several chronic myelogenous leukemia cell lines and transfectants, with IC(50) values in the low nanomolar range, 1 to 2 logs lower than those obtained with imatinib. Cells expressing activated forms of KIT or platelet-derived growth factor receptor (PDGFR), two additional targets of imatinib, were unaffected by SKI-606, whereas activity was found against PIM2. SKI-606 retained activity in cells where resistance to imatinib was caused by BCR-ABL gene amplification and in three of four Bcr-Abl point mutants tested. In vivo experiments confirmed SKI-606 activity in models where resistance was not caused by mutations as well as in cells carrying the Y253F, E255K, and D276G mutations. Modeling considerations attribute the superior activity of SKI-606 to its ability to bind a conformation of Bcr-Abl different from imatinib.

A Src/Abl Kinase Inhibitor, SKI-606, Blocks Breast Cancer Invasion, Growth, and Metastasis In vitro and In vivo

The central role of Src in the development of several malignancies, including breast cancer, and the accumulating evidence of its interaction with receptor tyrosine kinases, integrins, and steroid receptors have identified it as an attractive therapeutic target. In the current study, we have evaluated the effect of a Src/Abl kinase inhibitor, SKI-606, on breast cancer growth, migration, invasion, and metastasis. Treatment of human breast cancer cells MDA-MB-231 with SKI-606 caused a marked inhibition of cell proliferation, invasion, and migration by inhibiting mitogen-activated protein kinase and Akt phosphorylation. For in vivo studies, MDA-MB-231 cells transfected with the plasmid encoding green fluorescent protein (GFP; MDA-MB-231-GFP) were inoculated into the mammary fat pads of female BALB/c nu/nu mice. Once tumor volume reached 30 to 50 mm(3), animals were randomized and treated with vehicle alone or 150 mg/kg SKI-606 by daily oral gavage. Experimental animals receiving SKI-606 developed tumors of significantly smaller volume (45-54%) compared with control animals receiving vehicle alone. Analysis of lungs, liver, and spleen of these animals showed a significant decrease in GFP-positive tumor metastasis in animals receiving SKI-606 at a dose that was well tolerated. Western blot analysis and immunohistochemical analysis of primary tumors showed that these effects were due to the ability of SKI-606 to block tumor cell proliferation, angiogenesis, growth factor expression, and inhibition of Src-mediated signaling pathways in vivo. Together, the results from these studies provide compelling evidence for the role of Src inhibitors as therapeutic agents for blocking breast cancer growth and metastasis.

SKI-606, a Src/Abl Inhibitor with In vivo Activity in Colon Tumor Xenograft Models

Src up-regulation is a common event in human cancers. In colorectal cancer, increased Src levels are an indicator of poor prognosis, and progression to metastatic disease is associated with substantial increases in Src activity. Therefore, we examined the activity of SKI-606, a potent inhibitor of Src and Abl kinases, against colon tumor lines in vitro and in s.c. tumor xenograft models. SKI-606 inhibited Src autophosphorylation with an IC(50) of approximately 0.25 micromol/L in HT29 cells. Phosphorylation of Tyr(925) of focal adhesion kinase, a Src substrate, was reduced by similar concentrations of inhibitor. Antiproliferative activity on plastic did not correlate with Src inhibition in either HT29 or Colo205 cells (IC(50)s, 1.5 and 2.5 micromol/L, respectively), although submicromolar concentrations of SKI-606 inhibited HT29 cell colony formation in soft agar. SKI-606 also caused loosely aggregated Colo205 spheroids to condense into compact spheroids. On oral administration to nude mice at the lowest efficacious dose, peak plasma concentrations of approximately 3 micromol/L, an oral bioavailability of 18%, and a t(1/2) of 8.6 hours were observed. SKI-606 was orally active in s.c. colon tumor xenograft models and caused substantial reductions in Src autophosphorylation on Tyr(418) in HT29 and Colo205 tumors. SKI-606 inhibited HT29 tumor growth on once daily administration, whereas twice daily administration was necessary to inhibit Colo205, HCT116, and DLD1 tumor growth. These results support development of SKI-606 as a therapeutic agent for treatment of colorectal cancer.

Inhibitors of Src tyrosine kinase: the preparation and structure–activity relationship of 4-anilino-3-cyanoquinolines and 4-anilinoquinazolines

Src is a nonreceptor tyrosine kinase involved in signaling pathways that control proliferation, migration, and angiogenesis. Increased Src expression and activity are associated with an increase in tumor malignancy and poor prognosis. Several quinolines and quinazolines were identified as potent and selective inhibitors of Src kinase activity.

Discovery of benzisoxazoles as potent inhibitors of chaperone heat shock protein 90.

Heat shock protein 90 (Hsp90) is a molecular chaperone that is responsible for activating many signaling proteins and is a promising target in tumor biology. We have identified small-molecule benzisoxazole derivatives as Hsp90 inhibitors. Crystallographic studies show that these compounds bind in the ATP binding pocket interacting with the Asp93. Structure based optimization led to the identification of potent analogues, such as 13, with good biochemical profiles.

SKI-606 (Bosutinib) Blocks Prostate Cancer Invasion, Growth, and Metastasis In vitro and In vivo through Regulation of Genes Involved in Cancer Growth and Skeletal Metastasis

In the current study, we have examined the efficacy of a Src/Abl kinase inhibitor SKI-606 (Bosutinib) for its effect on prostate cancer growth and skeletal metastasis. Treatment of highly invasive human prostate cancer cells PC-3 and DU-145 with different doses of SKI-606 decreased Src activation, cell proliferation, migration, and invasion as determined by Matrigel Boyden chamber invasion assay. For in vivo studies, PC-3 cells were inoculated through s.c. or i.t. route into male BALB/c nu/nu or Fox Chase severe combined immunodeficient mice, respectively. Experimental animals treated with SKI-606 developed tumors of a significantly smaller volume and a significant decrease (50%) in experimental skeletal lesion area. A marked increase (32%) in bone volume to tumor volume ratio was also seen by micro–computed tomography analysis of tibias from control and experimental groups of animals. Western blot analysis showed the ability of SKI-606 to significantly decrease the phosphorylation of signaling molecules (AKT, mitogen-activated protein kinase, focal adhesion kinase) and the expression of tumor progression–associated genes uPAR, MMP-2, MMP-9, N-cadherin, fibronectin, BMP-2 (bone morphogenetic protein 2), BMP-6 (bone morphogenetic protein 6), IL-8 (interleukin 8), and TGF-β (transforming growth factor β) in prostate cancer cells. SKI-606 is currently in clinical trials for breast cancer and chronic myelogenous leukemia. Results from these studies provide convincing evidence for evaluating its efficacy in prostate cancer patients. Mol Cancer Ther; 9(5); 1147–57. ©2010 AACR.

Identification of a Conserved Sequence Motif That Promotes Cdc37 and Cyclin D1 Binding to Cdk4

Cdc37 is a molecular chaperone that is important for the stability and activity of several protein kinases, including Cdk4 and Raf1. We first determined, using in vitro assays, that Cdc37 binds to the amino-terminal lobe of Cdk4. Subsequent mutagenesis revealed that Gly-15 (G15A) and Gly-18 (G18A) were critical for Cdc37-Cdk4 complex formation. Gly-15 and Gly-18 of Cdk4 are within the conserved Gly-X-Gly-X-X-Gly motif that is required for ATP binding to the kinase. Mutation of either glycine at the equivalent positions of Raf1 (G358A and G361A) also inhibited Cdc37 binding to Raf1. Replacing another conserved residue critical for ATP binding and kinase activity, Lys-35 (K35A), reduced Cdc37-Cdk4 complex formation but to a lesser extent. The interaction of Cdk4 with Cdc37 in vitro was not sensitive to changes in ATP levels. Cell-based assays indicated that Cdk4G15A and Cdk4G18A were present at the same level as wild type Cdk4. Equivalent amounts of p16 bound to Cdk4G15A and Cdk4G18A relative to wild type Cdk4, suggesting that Cdk4G15A and Cdk4G18A adopt significant tertiary structure. However, in contrast to wild type Cdk4, Cdk4G15A, and Cdk4G18A had greatly reduced binding of cyclin D1, Cdc37, and Hsp90. Importantly, overexpression of Cdc37 not only stimulated cyclin D1 binding to wild type Cdk4 but also restored its binding to Cdk4G15A. Under the same conditions, p16 binding to wild type Cdk4 was suppressed. Our findings show that the interaction of Cdc37 with its client protein kinases requires amino acid residues within a motif that is present in many protein kinases.

Bosutinib: a review of preclinical studies in chronic myelogenous leukaemia.

Bosutinib (SKI-606) is an orally active Src and Abl kinase inhibitor presently in Phase III trials for treatment of chronic myelogenous leukaemia (CML), and in Phase II trials for treatment of breast cancer. Bosutinib is a potent antiproliferative and proapoptotic agent in CML cells and inhibits Bcr-Abl mediated signalling at nanomolar concentrations. Short-term administration of bosutinib causes regression of K562 and KU812 CML tumour xenografts. BaF3 murine myeloid cells expressing wild-type Bcr-Abl are sensitive to bosutinib treatment, as are BaF3 cells expressing many imatinib-resistant forms of Bcr-Abl. Recent studies indicate that bosutinib is active against a broader spectrum of kinases than originally believed. These additional inhibitory activities have interesting possibilities for further clinical development. This review will focus on preclinical studies supporting the clinical development of bosutinib for treatment of CML, with a discussion on the broader potential of this agent in other oncology indications.

Inhibition of Src kinase activity by 4-anilino-7-thienyl-3-quinolinecarbonitriles

Based on a screening lead from a yeast-based assay to identify Src family kinase inhibitors, a series of 4-anilino-7-thienyl-3-quinolinecarbonitriles was prepared. When the thiophene ring was substituted with a water-solubilizing group in a 2,5-, 3,5- or 2,4-pattern, potent inhibition of Src kinase activity was observed.

Substituted 4-anilino-7-phenyl-3-quinolinecarbonitriles as Src kinase inhibitors

A series of substituted 4-anilino-7-phenyl-3-quinolinecarbonitriles has been prepared as Src kinase inhibitors. Optimal activity is observed with compounds that have basic amines attached via the para position of the 7-phenyl ring, and a hydrogen atom at the C-6 position. The best compounds are low nanomolar inhibitors of Src kinase, and have potent activity against Src-transformed fibroblast cells.