Yi Zheng (ed.), Rational Drug Design: Methods and Protocols, Methods in Molecular Biology, vol. 928,
DOI 10.1007/978-1-62703-008-3_14, © Springer Science+Business Media New York 2012
An In Vitro Screening to Identify Drug-Resistant Mutations
for Target-Directed Chemotherapeutic Agents
The discovery of oncogenes and tumor suppressors as a driver of cancer development has triggered the
development of target-speci fi c small molecule anticancer compounds. As exempli fi ed by Imatinib (Gleevec),
a speci fi c 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. De fi ning the
spectrum of such mutations is important for patient monitoring and the design of next-generation inhibi-
tors. 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.
Key words: Protein kinase , Small molecule inhibitors , Tyrosine kinase inhibitors , Acquired drug
resistance , Retroviral-screening
Protein kinases are critical components of signaling networks that
catalyze protein phosphorylation and thereby regulate a wide vari-
ety of cellular functions including cell proliferation and death, cell
adhesion and motility, metabolism, transcription, and differentiation.
Spatiotemporal control of phosphorylation is crucial to homeosta-
sis and development, and this control relies on proper regulation of
kinases and phosphatases ( 1– 3 ) . An imbalance of phosphorylation/
dephosphorylation dynamics can have disastrous consequences for
cellular homeostasis often leading to cell transformation, cancer, or
a host of other diseases ( 4 ) . More than 400 human diseases have
been linked, directly or indirectly, to protein kinases ( 5 ) . Therefore,
the ability to modulate kinase activity represents an attractive
176 M. Azam
therapeutic intervention for the treatment of various human
illnesses ( 6– 8 ) .
Novel discoveries in cancer biology have provided the oppor-
tunity to design target-speci fi c anticancer agents and fostered
rapid advancements in drug development. The current focus is the
design of molecules with high selectivity against speci fi c proteins
in malignant cells to insure a high therapeutic index with minimal
side effects. The unprecedented success of the BCR/ABL tyrosine
kinase inhibitor Imatinib (Gleevec) in the treatment of chronic
myeloid leukemia (CML) has inspired great expectations for this
approach ( 9, 10 ) . Complete hematologic responses to Imatinib
are seen in >95% of CML patients and a major cytogenetic response
in >60% of patients treated in the chronic phase of the disease ( 7 ) .
However, highly speci fi c protein inhibition brings with it a critical
problem: protein targets develop escape mutations leading to drug
resistance. In fact, virtually all patients with advanced stages of
CML ultimately manifest Imatinib resistance ( 11– 13 ) , and it was
expected that other protein targets would evolve drug-resistant
forms as well in response to therapy. Indeed, pharmacological
inhibition of EGFR, c-KIT receptor, and PDGFR-alpha devel-
oped resistance to therapy by acquiring point mutations that
directly or indirectly affect drug binding, viz., drug resistance.
The identi fi cation of these mutant forms is essential for the design
of more robust next-generation therapies, and may ultimately lead
to molecular cocktails designed to circumvent resistance.
In previous work, we have reported the results of our screen
involving random mutagenesis of BCR/ABL to reveal the spec-
trum of mutations conferring resistance to BCR/ABL ( 14, 15 )
inhibitors such as Imatinib, PD166326 ( 15 ) , and AP4163 ( 16 ) .
The results not only identi fi ed the mutants critical for clinical dis-
ease relapse but also shed light on the structural regulatory mecha-
nisms of kinases ( 14, 17 ) . Here we provide additional methodologic
detail to enable a broader application of this screening strategy to
additional drug–target pairs.
1. pEYK3.1 or POP-PURO-Ires-GFP retroviral vectors for target
2. pCL-ECO ecotropic helper plasmid for the generation of
1. TOP10 Electrocompetent cells.
2. TOP10 Chemical competent cells.
2.2. E. coli Strains,
Reagents for Microbial
Culture, and Selection
177 14 An In Vitro Screening to Identify Drug-Resistant Mutations…
4. LB media.
5. LB agar.
6. XL-1 red competent cells (Agilent technologies, cat. # 200129).
1. 293T cells (American Type Culture Collection, cat. #
2. BAF3 cell (DSMZ# ACC 300).
3. Heat-inactivated FBS.
4. Penicillin/streptomycin solution 100×.
5. 70% (vol/vol) ethanol.
6. PBS without Ca/Mg.
9. WEHI conditioned media.
1. Protamine sulfate.
2. FuGENE 6 transfection reagent.
1. PCRXL-TOPO Kit.
2. Rapid DNA ligation kit.
3. EXPAND Long template PCR.
4. QUICKCHANGE XL mutagenesis kit.
1. Protease inhibitor cocktail from Roche.
2. SuperSignal West Pico Chemiluminescent Substrate.
3. Phosphatase Inhibitor Cocktail 1 (Sigma; cat. # P2850).
4. Phosphatase Inhibitor Cocktail 1 (Sigma; cat. # P5726).
5. Carnation Nonfat dry milk.
1. PCR Thermal cycler.
2. DNA gel electrophoresis system.
3. Protein gel electrophoresis system.
4. Protein gel transfer apparatus.
2.3. Cell Lines
2.4. Cell Culture Media
2.5. Reagents for DNA
Transfection and Viral
2.7. Reagents for DNA
for Protein Analysis
for Cell Culture, DNA,
and Protein Analysis
178 M. Azam
6. 37 C incubator shaker for bacterial culture.
7. Inverted tissue culture microscope with phase contrast (4, 10,
20, 40 objectives).
9. Biosafety cabinet with aspirator for tissue culture.
10. CO2 incubator, 37°C, humidity.
11. Tissue culture centrifuge.
12. Tissue culture dish, 35, 100, and 150 mm.
13. Tissue culture plates, 6 well, 12 well, and 96 well.
14. Conical tubes, 15 and 50 ml.
15. Glass Pasteur pipettes, 9 in., sterilized using autoclave.
16. Cryovials, 2.0 ml.
17. Plastic disposable transfer pipettes, 1, 5, 10, 25, and 50 ml.
18. Disposable sterile fi lter system, 0.22 m, 500 ml.
19. Disposable syringes, 10, 5, and 1 ml.
20. Hypodermic needle, 27–30 G.
21. Acrodisc fi lter, 0.45 mM, low protein binding.
22. Acrodisc fi lter, 0.2 mM, DMSO safe.
23. Sterile Petri dishes.
24. Coulter counter (Beckman Coulter) or hemocytometer.
To identify a wide spectrum of drug resistance conferring mutations,
we generated a high-complexity library of mutagenized BCR–ABL
cDNA in a retroviral vector and introduced this into cells by retro-
viral transduction. We then selected for surviving drug-resistant
clones, recovered plasmid DNA or PCR amplicons, and analyzed
their sequence for the presence of mutations. To verify that the
observed mutations were the basis of drug resistance, we regener-
ated each mutation separately by site-directed mutagenesis (SDM)
of native BCR–ABL, and reintroduced them into cells. Brie fl y, the
methodology is as described earlier (Fig. 1 ) ( 14, 18 ) ; clone the
target cDNA into a retroviral vector. Propagate the vector in bac-
teria de fi cient in DNA repair mechanisms, creating an exhaustive
library of mutations in the target gene. Transfect/infect drug-
sensitive cells with the mutated vector and disperse in soft agar in
the presence of drug. Isolate resistant colonies, recover the target
cDNA, and sequence to identify mutations. The resistant phenotype
179 14 An In Vitro Screening to Identify Drug-Resistant Mutations…
Fig. 1. A general scheme for drug-resistant screening of a drug–target pair.
180 M. Azam
of the mutations is then con fi rmed recreating the mutation in the
native cDNA by SDM. Cells are transfected/infected and grown in
the presence of drug. Resistance is measured by proliferation assays
and/or immunoblotting. Additionally, to gain insight about the
structural consequences on drug binding and resistance, mutations
are mapped on a model of the protein crystal structure to perform
in silico analysis.
1. Clone the desired target gene in either pEYK3.1 ( 19 ) or pOP-
puro-Ires GFP vector ( 17 ) (see Note 1 ).
2. Transform the recombinant clone to XL-1 red E. coli cells.
Thaw 100 m l XL-1 Red competent E-coli (Stratagene) in a
polypropylene tube on ice. Gently mix in 10–50 ng plasmid
DNA. Incubate on ice for 30 min, gently swirling every 2 min.
Immerse the tube in a 42°C bath for 45 s and immediately
incubate on ice for 2 min. Add 1 ml SOC medium and incu-
bate at 37°C shaking at 225–250 rpm for 90 min.
3. Plate the transformation mix on LB-agar plate containing zeocin
(25 ug/ml) and incubate at 37°C for 36–48 h (see Note 2).
4. Collect the colonies by scraping the plates with a sterile plate
scraper. Isolate plasmid DNA using the Qiagen midi-prep Kit.
At this stage, the heterogeneity of mutations in the library can
be roughly assessed by restriction digestion with a frequent
cutter such as Sau3A1 or Taq1 or by DNA sequencing .
1. One day before transfection, plate 2 × 10 6 HEK 293 T cells
onto six 100 mm dishes in DMEM containing 10% FCS, pen/
strep, and 2 mM L -glutamine.
2. Replace the medium the next day, and transfect the cells with
mutagenized library of target gene. Transfect each 100 mm
plate with 10 m g of DNA (mix 5 m g of mutagenized retroviral
library with 5 m g of retroviral packaging construct pCL-eco 8
and then add 30 m l of FuGENE6).
3. Pour the transformation mix dropwise on the HEK293T cells.
Incubate the plate overnight at 37 C.
4. Change media the next day, and carefully add 10 ml of medium
over the cells as they are loosely attached to the surface.
5. Collect the media after 48 h containing retroviruses. Filter the
viral supernatant through a 0.45 m m Acrodisc fi lter to remove
cell debris and particulate matters. The retroviruses can be
snap frozen with liquid nitrogen and stored at −80°C or used
immediately for transduction experiments.
1. Grow BaF3 cells in R10 media (RPMI with 10% FCS, 100 U
Penicillin/100 microgram Streptomycin per ml, 2 mM
L -glutamine, and 10% WEHI-3B conditioned medium (as a
Construct and Random
18114 An In Vitro Screening to Identify Drug-Resistant Mutations…
source of IL-3) . The BaF3 cell line is a murine pro B cell line
that is dependent on Interleukin-3 (IL-3) for growth. Ectopic
expression of constitutively activated tyrosine kinases such as
BCR–ABL, EGFR, SRC, PDGFR, and JAK2-V617F renders
the BaF3 cells IL-3 independent 10 .
2. Transfer 1 × 10 6 BaF3 cells in each well of 6-well tissue culture
3. Add 1 ml of viral supernatant having a titer of 10 5 –10 6 infec-
tious units/ml (see Note 3 ).
4. Add 3 m l of polyberene (8 mg/ml) and 1 ml of R10 media
5. Centrifuge the plate for 90 min at 1,500×g in a Sorvall RT
6000 table centrifuge.
6. Transfer the plates to the incubator for 14–16 h at 37°C with
5% CO 2 (see Note 4 ).
1. To select for drug-resistant clones, plate the virally transduced
cells in soft agar containing varying concentrations of drugs.
Mix 8 × 10 6 cells with 28.8 ml RPMI, 9.6 ml FCS, 9.6 ml of
1.2% Bacto-agar (made in PBS, autoclaved, and cooled to
42°C), and supplemented with varying concentrations of drugs
(1–20 m M).
2. Plate 3 ml of cell-mix per well into 6-well plates and incubate
at 37°C, 5% CO 2 , for 14–24 days.
3. Pick the single colonies and expand separately in 3 ml R10
media in the presence of drugs.
4. Collect the cells after reaching to con fl uence. Isolate the
genomic DNA using the Qiagen DNeasy Kit.
5. Target DNA can be sequenced either by rescuing the whole
gene followed by subcloning or by sequencing PCR amplicons
of desired region of the target gene (see Note 5 ).
1. Digest 10 m g of genomic DNA with NotI to release the
2. Ligate the digested DNA (100 m l of reaction volume per 1 m g
of genomic DNA) using T4 DNA ligase from Roche.
3. Extract the DNA using phenol/chloroform extraction and
4. Resuspend the precipitated DNA in 10 ml of water. Transform
the TOP10 competent cells (Invitrogen) with ligated DNA.
5. Plate the transformation mix on LB-agar/zeocin plates.
6. Pick the colonies, and isolate the plasmid DNA using Qiagen
mini-prep kit for sequencing (see Note 6 ).
for Resistant Clones
3.5. Provirus Rescue
from Genomic DNA
182 M. Azam
1. To speci fi cally mutate a desired residue we performed SDM on
our plasmids using the Quickchange Mutagenesis Kit from
Stratagene and oligonucleotides that were designed to create
the point mutations found in our screen. Con fi rm the muta-
tions by DNA sequencing (see Note 7 ).
1. Plate 10 4 BaF3 cells expressing mutant genes into each well of
a 96-well plate in RPMI/10% FCS.
2. Add the drugs to the media in increasing concentrations ( fi nal
concentration: 0, 1, 3, 5, 10, and 20 m M) across the plate.
3. Incubate the plate for 48–60 h at 37°C, 5% CO 2 .
4. Assess the cell viability by adding 10 ml of WST-1 reagent
5. Read the plate with an ELISA plate reader at 450 nm (see
Note 8 ).
1. We recommend using either pEYK3.1 ( 19 ) or pOP-puro-Ires-
GFP ( 17 ) vectors for screening because they have single LTR
with an engineered Not I site, which facilitates the proviral
rescue from genomic DNA. Additionally, single LTR-based
vector can be used directly for SDM, as conventional retroviral
vectors having two LTRs are not good substrate for SDM reac-
tion because they tend to loop out the inserts during PCR
ampli fi cation.
2. Because XL-1 red cells are growing cells, colonies start appear-
ing after 24 h. We recommend harvesting the cells as soon as
colonies start appearing (typically 24–36 h but in some cases it
may take a little longer depending on the type of genes) fol-
lowed by plasmid miniprep. Use this puri fi ed DNA to trans-
form the normal XL-1 blue E. coli -competent cells to amplify
the plasmid library.
3. We have employed low viral transduction ef fi ciency (e.g.,
20–30%) in order to avoid inducing a drug-resistant pheno-
type due to multiple integration of provirus. Therefore we rec-
ommend not to use high-titer viral supernatant or overexpression
of the protein to a speci fi c level.
4. During our screen, we found that it is important to avoid bulk
culture conditions in which cells harboring different mutations
are pooled together and allowed to expand in liquid culture,
since this can lead to clonal dominance of a few highly drug-
resistant variants. When we initially selected for imatinib
3.7. Cell Viability
Assay to Determine
18314 An In Vitro Screening to Identify Drug-Resistant Mutations…
resistance of mutagenized BCR/ABL in bulk liquid culture,
we found only 4 mutant forms that were represented multiple
times within the fi rst 100 isolates we sequenced. In contrast,
we identi fi ed over 100 mutants when we selected for out-
growth of clones in the soft agar colony-forming assay. Some
slow-growing clones with more modest degrees of drug
resistance could not have been recovered using bulk culture.
We thus recommend to avoid any growth or selection in bulk,
within either the bacterial or mammalian cell cultures. For that
reason, we attempted to minimize any incubation periods
where cells are pooled together. After retroviral infection, we
maintained the cells in bulk culture prior to selection for only
14–16 h, after which we performed combined IL-3 withdrawal
and drug selection in soft agar. This provided for tight selec-
tion of individual clones and prevented clonal dominance of
liquid cultures. If the cells used for drug screening do not form
colonies in soft agar they can be selected in a similar clonal
manner by limiting dilution in 96-well plates.
5. We recommend using PCR ampli fi cation of the desired region
in a given gene but in some instances we failed to amplify the
desired target due to complexities incurred by genomic DNAs.
In such cases we performed the proviral rescue to clone whole
6. Proviruses can be rescued by Not I digestion followed by
ligation/transformation. However, in some cases transgenes
have an internal Not I site, e.g., SRC kinase; in this situation
we used Cre recombinase mediate excision of proviruses by
incubating the genomic DNA with recombinant Cre for
30 min at 37 C followed by transformation plating on zeocin
plate. In pEYK3.1 and pOP-puro-Ires-GFP vectors, we have
engineered Cre recombination site fl anking the LTR. Because
of low ef fi ciency of Cre recombination as compared to Not I
digestion, we do not recommend to use Cre-mediated provi-
rus rescue as a fi rst choice but it can be used in the cases when
the transgenes are harboring internal Not I sites.
7. We recommend using freshly prepared plasmids as a substrate
for SDM reaction. Older plasmid preparation or nicked plas-
mids give lots of background and often fail to produce the
desired mutant clones.
8. All assays should be performed in quadruplicate and readings
are averaged and plotted against drug concentration as a
best- fi t sigmoidal curve using a nonlinear curve- fi tting algo-
rithm (Origin 7.0, Origin Lab, Northampton, MA). The
drug concentration resulting in 50% cell viability scored as
the Cellular IC50.
184 M. Azam
This work was supported by grants from V Foundation.
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