Abstract
The discovery of oncogenes and tumor suppressors as a driver of cancer development has triggered the development of target-specific small molecule anticancer compounds. As exemplified by Imatinib (Gleevec), a specific inhibitor of the Chronic Myeloid Leukemia-associated BCR/ABL kinase, these agents promise impressive activity in clinical trials, with low levels of clinical toxicity. However, such therapy is susceptible to the emergence of drug resistance mainly due to amino acid substitutions in the target protein. Defining the spectrum of such mutations is important for patient monitoring and the design of next-generation inhibitors. Using Imatinib and BCR/ABL as a paradigm for a drug–target pair, we reported a retroviral vector-based screening strategy to identify the spectrum of resistance-conferring mutations, which has helped in designing the next-generation BCR/ABL inhibitors such as Nilotinib, Dasatinib, and Ponatinib. Here we provide a detailed methodology for the screen, which can be generally applied to any drug–target pair.
This is a preview of subscription content, log in via an institution.
Buying options
Tax calculation will be finalised at checkout
Purchases are for personal use only
Learn about institutional subscriptionsReferences
Azam M, Daley GQ (2006) Anticipating clinical resistance to target-directed agents: the BCR-ABL paradigm. Mol Diagn Ther 10:67–76
Hanahan D, Weinberg RA (2000) The hallmarks of cancer. Cell 100:57–70
Blume-Jensen P, Hunter T (2001) Oncogenic kinase signalling. Nature 411:355–65
Huse M, Kuriyan J (2002) The conformational plasticity of protein kinases. Cell 109:275–82
Melnikova I, Golden J (2004) Targeting protein kinases. Nat Rev Drug Discov 3:993–4
Cohen P (2002) Protein kinases–the major drug targets of the twenty-first century? Nat Rev Drug Discov 1:309–15
Dancey J, Sausville EA (2003) Issues and progress with protein kinase inhibitors for cancer treatment. Nat Rev Drug Discov 2:296–313
Noble ME, Endicott JA, Johnson LN (2004) Protein kinase inhibitors: insights into drug design from structure. Science 303:1800–5
Druker BJ, Sawyers CL, Kantarjian H, Resta DJ, Reese SF, Ford JM, Capdeville R, Talpaz M (2001) Activity of a specific inhibitor of the BCR-ABL tyrosine kinase in the blast crisis of chronic myeloid leukemia and acute lymphoblastic leukemia with the Philadelphia chromosome. N Engl J Med 344:1038–42
Druker BJ, Tamura S, Buchdunger E, Ohno S, Segal GM, Fanning S, Zimmermann J, Lydon NB (1996) Effects of a selective inhibitor of the Abl tyrosine kinase on the growth of Bcr-Abl positive cells. Nat Med 2:561–6
Gorre ME, Mohammed M, Ellwood K, Hsu N, Paquette R, Rao PN, Sawyers CL (2001) Clinical resistance to STI-571 cancer therapy caused by BCR-ABL gene mutation or amplification. Science 293:876–80
Shah NP, Nicoll JM, Nagar B, Gorre ME, Paquette RL, Kuriyan J, Sawyers CL (2002) Multiple BCR-ABL kinase domain mutations confer polyclonal resistance to the tyrosine kinase inhibitor imatinib (STI571) in chronic phase and blast crisis chronic myeloid leukemia. Cancer Cell 2:117–25
Branford S, Rudzki Z, Walsh S, Grigg A, Arthur C, Taylor K, Herrmann R, Lynch KP, Hughes TP (2002) High frequency of point mutations clustered within the adenosine triphosphate-binding region of BCR/ABL in patients with chronic myeloid leukemia or Ph-positive acute lymphoblastic leukemia who develop imatinib (STI571) resistance. Blood 99:3472–5
Azam M, Latek RR, Daley GQ (2003) Mechanisms of autoinhibition and STI-571/imatinib resistance revealed by mutagenesis of BCR-ABL. Cell 112:831–43
Azam M, Nardi V, Shakespeare WC, Metcalf CA 3rd, Bohacek RS, Wang Y, Sundaramoorthi R, Sliz P, Veach DR, Bornmann WG, Clarkson B, Dalgarno DC, Sawyer TK, Daley GQ (2006) Activity of dual SRC-ABL inhibitors highlights the role of BCR/ABL kinase dynamics in drug resistance. Proc Natl Acad Sci USA 103:9244–9
Azam M, Powers JT, Einhorn W, Huang WS, Shakespeare WC, Zhu X, Dalgarno D, Clackson T, Sawyer TK, Daley GQ (2010) AP24163 inhibits the gatekeeper mutant of BCR-ABL and suppresses in vitro resistance. Chem Biol Drug Des 75:223–7
Azam M, Seeliger MA, Gray NS, Kuriyan J, Daley GQ (2008) Activation of tyrosine kinases by mutation of the gatekeeper threonine. Nat Struct Mol Biol 15:1109–18
Azam M, Raz T, Nardi V, Opitz SL, Daley GQ (2003) A screen to identify drug resistant variants to target-directed anti-cancer agents. Biol Proced Online 5:204–10
Koh EY, Chen T, Daley GQ (2002) Novel retroviral vectors to facilitate expression screens in mammalian cells. Nucleic Acids Res 30:e142
Acknowledgment
This work was supported by grants from V Foundation.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2012 Springer Science+Business Media New York
About this protocol
Cite this protocol
Azam, M. (2012). An In Vitro Screening to Identify Drug-Resistant Mutations for Target-Directed Chemotherapeutic Agents. In: Zheng, Y. (eds) Rational Drug Design. Methods in Molecular Biology, vol 928. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-008-3_14
Download citation
DOI: https://doi.org/10.1007/978-1-62703-008-3_14
Published:
Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-1-62703-007-6
Online ISBN: 978-1-62703-008-3
eBook Packages: Springer Protocols