[Frontiers in Bioscience E3, 1273-1288, June 1, 2011]

NPB001-05 inhibits Bcr-Abl kinase leading to apoptosis of imatinib-resistant cells

Vilas Wagh1, Shailaja Chile1, Sonal Monahar1, Bikas Chandra Pal2, Santu Bandyopadhyay3, Somesh Sharma1, Kalpana Joshi1

1Department of Pharmacology, Piramal Life Sciences Limited, 1-Nirlon complex, Goregaon, 400063 Mumbai, India, 2Chemistry Division, 3Division of Infectious Diseases and Immunology, Indian Institute of Chemical Biology (IICB, CSIR), 700032 Kolkata, India

TABLE OF CONTENTS

1. Abstract
2. Introduction
3. Materials and methods
3.1. Plant material, extraction and purification
3.2. Reagents
3.3. Cell lines
3.4. Clinical Samples
3.4. Cytotoxicity assay/proliferation assay
3.5. Cell cycle analysis apoptosis assay
3.6. In-vitro kinase assay
3.7. Data analysis
4. Results
4.1. Discovery and preparation of NPB001-05
4.2. Effect of NPB001-05 on growth of Bcr-Abl positive and negative cells, primary cultures and normal human peripheral blood mononuclear cells (PBMCs)
4.3. NPB001-05 inhibits proliferation of leukemic cell lines independent of Bcr-Abl mutational status
4.4. NPB001-05 inhibits autophosphorylation of wt and resistant Bcr-Abl
4.5. Effect of NPB001-05 on CML clinical samples
4.6. NPB001-05 blocks cell cycle progressing and induces apoptosis of Bcr-Abl positive cells
4.7. In-vitro profiling of NPB001-05 on different kinases
5. Discussion
6. Acknowledgements
7. References

1. ABSTRACT

The deregulated activity of the Bcr-Abl tyrosine kinase provides a rational basis for the development therapeutics in all phases of Chronic Myelogenous Leukemia (CML). Although a well studied imatinib therapy has clinical success against CML, resistance to imatinib due to mutations in the kinase domain, especially T315I poses a major problem for the ultimate success of CML therapy by this agent. Herein we describe an NPB001-05, derived from extract of Piper betle leafs, which is highly active in specifically inhibiting Bcr-Abl expressing cells. NPB001-05 inhibited the proliferation of BaF3 cells ectopically expressing wild type Bcr-Abl phenotype and 12 different imatinib-resistant mutations of clinical relevance (average IC50 5.7 µg/ml). Moreover, NPB001-05 was highly inhibitory to wild type P210Bcr-Abl and P210Bcr-Abl-T315I kinase activity and abrogated the autophosphorylating enzyme in time- and dose- dependent manner. NPB001-05 was non-toxic on normal cells, but was inhibitory to CML patient derived peripheral blood mononuclear cells. Treatment with NPB001-05 caused apoptosis induction and G0G1 cell cycle arrest in both Bcr-Abl wild type and T315I mutant cell lines.