Study of the interaction between Abl kinase domain and Gleevec inhibitor Authors: Alec Elder, Sarah Moser, Molly FieldsCHM 33901 Biochemsitry Laboratory Section: 1, Group: 2January 13, 2015 – April 28, 2015

AbstractGleevec, an important cancer treatment drug, inhibits the Abelson kinase domain to stop uncontrolled cell growth. In some patients the treatment has stopped working or had no effect at all. Gleevec cannot bind to an Abelson kinase (Abl) domain that has acquired mutations. This finding is very important for future cancer treatment, specifically in chronic myelogenous leukemia (CML). To study the effects of mutations in the Abelson kinase domain that effect Gleevec binding we used; PCR to amplify the mutation of interest, protein purification techniques, and Bradford/kinase assays to determine protein concentration and kinase activity respectively. Our results did support our hypothesis, but with small statistical significance; Gleevec will have less inhibition on the mutant Abl- S417Y compared with the wild type Abl, but due to impure protein samples of both WT and mutant Abl the concentration of target protein was uncertain.

Introduction

Tyrosine kinases are associated with signal transduction. One particular tyrosine kinase, Abelson (depicted below) is tightly regulated. It is auto-inhibited at its N-terminus and the default activity setting is off. Abl’s break cluster region (BCR), part from chromosome 9 and part from chromosome 22, results in a mutant gene called BCR-ABL. The protein it encodes, Bcr-Abl, includes the kinase domain of c-Abl but lacks the part responsible for auto-inhibition. The loss of the residues

responsible for auto-inhibition, results in the kinase being permanently on. This mutation is responsible for uncontrolled cell proliferation, which can lead to cancer.

Its inhibition has been a success in the treatment of chronic myelogenous leukemia. Abl Tyrosine kinase inhibitor imatinib, also known as Gleevec, works by inhibiting Bcr-ABL. It binds to the ATP-binding site and in the nearby hydrophobic

pocket in the inactive conformation of the Abl kinase domain (Taylor, 2010). Eventually, some CML patients develop a resistance to Gleevec and a few others do not respond to Gleevec at all. The resistance to Gleevec has been linked to mutations in the ABL kinase domain. A mutation would make the binding of Gleevec to the binding sight unstable. Over fifty different mutations, including single amino acid point mutations have been identified (Taylor,2010).

In this experiment we use Abl kinase as a model for the full-length Bcr- Abl protein. Abl kinase lacks the N-terminal Abl regulation domains and is therefore constitutively active. We expressed and purified WT Abl kinase domain and used site-directed mutagenesis to make a DNA expression vector for S417Y mutants. This mutant and H396P have been identified in patients with Gleevec resistance.

We determined whether S417Y mutation in the BCR-ABL gene confers Gleevec resistance using an in vitro kinase assay by observations in the kinase activity in Wild type Abl and S417Y mutant Abl domains, in the presence and absence of Gleevec inhibitor. We evaluate crystal structures to understand the mechanisms by which Bcr-Abl mutations block drug activity.

Methods

Primer designWe designed a primer for the Abl

kinase domain mutant S417Y for use in polymerase chain reaction by making a codon change in the middle of a 15-20 codon sequence. Our primer resides at the 674 bp to 698 bp of the wild type (WT) Abl sequence. The primer melting temperature was determined from the G-C base pair percentage content and a GC clamp applied to the 3’ end. The concentration of the

primers was determined by measuring the optical density at 260 nm on the UV spectrophotometer. Using the concentration found a 25μM stock solution of the primers was made to be used in the PCR reactions.

Polymerase Chain Reaction The polymerase chain reaction

(PCR) to make the chimera DNA was carried out in two reactions per mutant with both reactions containing deionized water, the pET28a-Abl plasmid and GoTaq Green Master mix (2x). In reaction one, T7 promoter primer (25μM) and mutant S417Y reverse primer (25μM) were added and in reaction two T7 terminator primer (25μM) and mutant S417Y forward primer (25μM) were added. These samples were placed in a thermocycler set to a 50OC annealing temperature and went through 30 cycles. After the cycles completed, the samples were run on a 1% agarose gel for analysis. Qiagen QIAquick PCR purification kit was used to purify the samples and a final PCR was run in a thermocycler at 45OC annealing temperature containing the two purified PCR reactions from above, deionized water, GoTaq Green Master Mix, T7 promoter primer (25μM) and T7 terminator primer (25μM) to make the full length S417Y mutant of 1114 bps. This final PCR reaction was analyzed on a 1% agarose gel and purified using the Qiagen QIAquick PCR purification kit.

Xbal and xhol enzyme digest An Xhol/Xbal double digest was

carried out by taking the final purified S417Y mutant, deionized water, 1X CutSmart Buffer and the Xhol/Xbal enzymes and incubated in a 37OC water bath. We applied the same digest to the vector using miniprep DNA, deionized water, 1X CutSmart Buffer and the Xhol/Xbal enzymes. The digests were run on a 1% agarose gel and the gel excised of

the insert S417Y mutant. The gel slices containing the mutant were purified using the Qiagen QIAquick Gel Extraction Kit.

Ligation and transformationLigation reaction included the

vector, insert, 2X Rapid Ligation buffer and T4 DNA ligase. A negative control was also ligated using vector, deionized water, 2X Rapid Ligation buffer and T4 DNA ligase. Transformation into E.coli DH5α plasmid was performed by using the ligation reactions and the pET28a-Abl WT plasmid. The mixture was incubated on ice, a heat shock applied and S.O.C (Super Optimal broth with Catabolite repression) media added to each tube. Using sterile technique the transformed cells were spread onto agar plates and incubated at 37OC overnight and re-streaked. For transformation of the S417Y mutant we combined the mutant and YopH plasmid to BL21DE3 cells and performed the same transformation procedure as the WT from there.

Isolation of S417Y Abl mutant plasmid

Qiagen QIAquick Miniprep Kit used to isolate the plasmid DNA of the S417Y Abl mutants. Another PCR carried out to confirm the mutation in S417Y Abl with a forward reaction containing miniprep DNA, T7 promoter primer (0.8 μM), Big Dye 3.1 Polymerase MIx and deionized water, and a reverse reaction containing miniprep DNA, T7 terminator primer (0.8 μM), Big Dye 3.1 Polymerase Mix and deionized water. The thermocycler was set to a 45OC annealing temperature for 99 cycles.

Expression of Abl kinase in presence of Yop phosphatase

LB/kan/strep was inoculated with the culture of Abl and Yop bacteria and placed in a 37OC shaker, while the optical density was checked periodically at 600 nm until it

reached 1.0 OD. A sample was collected for our SDS-PAGE analysis. IPTG (isopropylthio-β-galactosidase) was added to induce the protein overnight.

Buffer PreparationNi-affinity purification column

buffers: Ni-NTA binding buffer (50 mM Tris, 300 mM NaCl, pH 7.8); Ni-NTA washing (50 mM Tris, 300 mM NaCl, 30 mM imidazole, pH 7.8); and Ni-NTA elution buffer (50 mM Tris 300 mM NaCl, 30 mM imidazole, pH 7.8). Dialysis buffer (10X Tris-buffered saline (TBS) dialysis buffer (200 mM Tris, 1.37 M NaCl, pH 7.5). SDS-PAGE buffers: 1.5 M Tris-HCl (pH 8.8); 0.5 M Tris-HCl (pH 6.8); 10X Tris-Glycine-SDS electrophoresis buffer made using 20% SDS; and 10X Tris-Glycine (TG) transfer buffer. 5X reducing protein loading buffer (SDS sample buffer), Coomassie staining solution (0.25% Coomassie Brilliant Blue, 50% methanol, and 10% glacial acetic acid), Fast destain solution (40% methanol and 10% glacial acetic acid), Slow destain solution (5% methanol and 10% glacial acetic acid), 10% ammonium persulfate (APS), 40% acrylamide/bisacrylamide solution, 20% SDS solution, and TEMED (tetramethylethylenediamine).

Cell lysis and isolation using Ni-NTA resin purification

The WT Abl cells were lysed using B-PER detergent and protease inhibitor cocktail solution until homogenous. Purification of the WT Abl domain using Ni-NTA resin by affinity chromatography was carried out in three stages of Ni-NTA binding buffer added and collected, washing buffer added and collected, and lastly elution buffer added and collected. The remaining WT Abl protein from the purification was loaded into a dialysis cassette and left to dialyze in TBS at 4OC

overnight. This step was also performed for the S417Y mutant Abl.

Bradford AssayThe dialyzed solution was placed

into a 10kDa MWCO centrifugal concentrator and spun down to concentrated WT Abl protein. We quantified the protein using a Bradford Assay of the standard Bovine Serum Albumin. Coomassie Brilliant Blue reagent was added to view the protein samples in the spectrophotometer. This step was also performed for the S417Y mutant Abl.

SDS-PAGEPreparation of the purification

samples included adding 2X sample loading buffer to the samples to be run on the SDS-PAGE gel. We ran two SDS-PAGE gels for the WT and S417Y mutant both made with a 10% separating gel using deionized water, 1.5 M Tris-HCl (pH 8.8), 40% acrylamide/bisacrylamide solution and 10% SDS. The stacking gel contained deionized water, 0.5 M Tris-HCL (pH 6.8), 40 % acrylamide/bisacrylamide solution and 10% SDS. After running the gels, we stained them using Coomassie Brilliant Blue staining solution to aid in analysis.

Kinase assay with or without Gleevec present

A standard curve for the serial dilutions was made using 1X assay buffer and 1mM stock phosphate in 8 tubes and doubled. The samples were prepared by making the solutions a 1 mM ATP stock

using 10 mM ATP and 1x assay buffer, 1 mM ADP stock using 10 mM ADP and 1x assay buffer, 10 ng/μL Coupling phosphatase 4 from 100 ng/μL CP4 and 1x assay buffer,0.33 μg/μL WT/S417Y mutant protein by adding the protein and 1X assay buffer, 0.75mM of peptide and 25 μM of Gleevec inhibitor. 2 tubes contained the negative control with ATP (0.2 mM), peptide substrate (0.2 mM), CP4 (0.2μg), 1x assay buffer, and DMSO. 2 tubes contained the positive control with ADP (0.2mM), peptide substrate (0.2 mM), CP4 (0.2μg), 1x assay buffer and DMSO. 2 tubes contained the WT Abl kinase - inhibitor with ATP (0.2 mM), peptide substrate (0.2 mM), CP4 (0.2μg), WT Abl (0.1 μg/μL) and DMSO. 2 tubes contained the WT Abl kinase + inhibitor with ATP (0.2 mM), peptide substrate (0.2 mM), CP4 (0.2μg), WT Abl (0.1 μg/μL) and Gleevec inhibitor (0.5μM). 2 tubes contained S417Y mutant Abl kinase - inhibitor with ATP (0.2 mM), peptide substrate (0.2 mM), CP4 (0.2μg), S417Y Abl (0.1 μg/μL) and DMSO. 2 tubes contained S417Y mutant Abl kinase + inhibitor with ATP (0.2 mM), peptide substrate (0.2 mM), CP4 (0.2μg), S417Y Abl (0.1 μg/μL) and Gleevec inhibitor (0.5μM). Malachite Green Reagents A and B were added to the tubes and the OD observed to make the standard curve for the kinase assay.

Crystal structure viewing and modeling

PyMol was used to view the crystal structure of Abl- S417Y and Gleevec.

Results

Fig. 1:Analysis of PCR reactions 1 & 2 on 1% Agarose gel for S417Y Abl mutants lane 4:GoTaq (not purified) PCR reaction 1: ~743 bp, lane 5: GoTaq (not purified) PCR reaction 2:~395 bpFig. 2 Final PCR reaction on 1% agarose gel Fig. 3: Xhol and Xbal double digest of Abl-S417Y mutants

Fig. 4: Transformation examples using Abl-H396P (example)Plate 1: Vector only negative control Plate 2: pET28a-ABL positive controlPlate 3: Abl-H396P

In figure one; we analyzed PCR reactions to see if our primer designs were successful. The band in lane four is estimated to be around 743 bp and the band in lane five to be about 395 bp. Reaction 1 contained the T7 promoter and mutant reverse primer whereas reaction 2 contained the T7 terminator and mutant forward primer. We then ran a final PCR reaction to analyze the full-length S417Y Abl mutant DNA using gel electrophoresis. In figure two you can see a bright band estimated to be around 1114 bp.

We then ran another gel electrophoresis with the S417Y mutant insert digest to excise the mutant gene and purify it by determining DNA concentration and purity. In figure 3 a band around 974 bp is present, which we then excised. The mutant inserts were then transformed using E.Coli DH5α competent cells for plasmid replication. In figure four the picture is of an example negative, positive and Abl-H396P were as expected. Our Abl-S417 mutant turned results were very similar.

Fig. 4

Fig. 1 Fig. 2Fig. 2Fig. 1

Fig. 6 Bradford assay for purified and concentrated WT Abl protein: Average to yield final concentration: 0.89 mg/ml, BSA stock concentration used: 0.51 µg/µLFig. 7 Bradford assay for purified and concentrated mutant Abl protein S417Y: Average to yield final concentration: 0.82 mg/ml

Fig. 7: Coomassie Stain of SDS-PAGE of WT Abl samplesLane 1 & 2: 2x SDS, lane 3: Standard, lane 4: pre-induction sample, lane 5: post-induction sample, lane 6: binding buffer sample, lane 7: wash buffer sample, lane 8: elution buffer sample, lane 9: pure sample, lane 10: 2x SFig. 8 Coomassie Stain of SDS-PAGE of Abl- S417Y samplesLane 1 & 2: 2x SDS, lane 3: Standard, lane 4: pre-induction sample, lane 5: post-induction sample, lane 6: binding buffer sample, lane 7: wash buffer sample, lane 8: elution buffer sample, lane 9: pure sample, lane 10: 2x SDS

We then did a Bradford assay to determine the final concentration of our purified and concentrated WT Abl protein, shown in figure 5. We then expressed the Abl-S417Y mutant kinase domains using IPTG induction. Figure 6 shows our Bradford assay standard curve which we used to find our Abl S417Y protein concentration. We then prepared an SDS-PAGE gel to check the purity of the WT Abl mutant kinase domain. Figure 7 shows the results of the SDS-PAGE. Lane 9 shows our protein between 37-25 D. We also have a

band just above 37 D and between 75 and 50 D. Next we did another SDS-PAGE with coomassie brilliant blue stain again, shown in figure 8. Lane 9 is the pure sample. The protein’s band is located between 25-37D, another band around 37 D, one around 50 and some smaller bands and streaks.

We did a kinase assay and found the standard curve which we then found the percent inhibition from, figures 10 and 9 respectively. Finally we used PyMol to view the crystal structure of Abl-S417Y mutant (Figures 11 through 13.)

Fig. 5 Fig. 6

1 2 3 4 5 6 7 8 9 10 1 2 3 4 5 6 7 8 9 10

Fig. 8Fig. 7

Fig. 11 Abl- S417Y crystal structureFig. 12 Stick structureAbl-S417Y binding pocket: magenta, Gleevec: green, DFG motif: blue, Binding A loop: yellowFig. 13 Cartoon and stick structureAbl-S417Y binding pocket: green, Gleevec: orange, DFG motif: Blue, Binding A loop: magenta

Discussion

In this experiment we investigated how mutations in Abl kinase could impose drug resistance to Gleevec, a common drug

for the treatment of CML. To do this we used WT Abl kinase along with an S417Y mutant Abl kinase.  We first needed to make this mutation using site-directed mutagenesis which was done using two step PCR.  pET28a-Abl plasmid was used along with mutant forward and reverse primers in

Fig. 13

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Fig. 13

Fig. 10

Fig. 9

Fig. 12

Fig. 11

separate reactions.  Reaction 1 contained the T7 promoter primer and Mutant Reverse primer while reaction 2 contained the T7 terminator primer and mutant forward primer.  These reactions would amplify certain sequences with predetermined length in order to verify the reaction accuracy.  The expected results included a 743 base pair (BP) segment from reaction 1 and a 395 BP segment from reaction 2.  An agarose gel was run and results are posted in figure 1. This experiment was successful according to the DNA ladder comparison. The lengths of target DNA were between the 850 BP and 650 BP markers for reaction 1 and between the 400 BP and 300 BP markers for reaction 2.

Next we needed to make a full length S417Y mutant.  To do this T7 terminator and promoters were used along with the purified DNA templates from reactions 1 and 2.  Figure 2 depicts the agarose gel from this experiment.  The sample was not pure but there was high fluorescence in the band which was slightly higher than the 1000 BP marker. The size of the full length S417Y mutant is 1114 BP, making the Abl-S417Y final PCR reaction a success.  This band was excised and the DNA extracted using a Qiagen purification kit.  Next restriction endonuclease digests were completed using Xbal and Xhol to create “sticky” ends on the final PCR products, gel electrophoresis was used to check the accuracy of the digests. Figure 3 displays this gel and confirms that the experiment was accurate resulting in a band close to the 1000BP marker which is close to the expected 974 BP.

This mutant insert was then ligated into an antibiotic resistant vector using T4 DNA ligase.  E. coli DH5 alpha cells were used to replicate the plasmid. We used a positive control (pET28a-Abl WT plasmid) which is a complete plasmid (no ligation reaction was performed) that has resistance to Kanamycin. The negative control did not

have the insert so the vector was in fragments, thus not having kanamycin resistance. Because not all ligation reactions were successful for the mutant, not as many colonies were seen on the plate as compared to the positive control.  The colonies that did grow on the treated plate therefore contained the insert and were selected for further growth as seen in figure 4.

Two colonies from the mutant cultures were chosen and grown in starter culture.  Mutant bacterial cells were also pelleted and the DNA was extracted from both colonies. The concentration and purity of the DNA was then checked using the 260/280 ratio before being sequenced by unidirectional PCR with fluorescently labeled ddNTPs to confirm the desired mutation and the absence of any undesirable mutations. Once the mutation was confirmed the mutant was co-expressed with YopH plasmid and was transformed into E. coli BL21DE3 cells. YopH plasmid was used because of its high specificity for phosphotyrosine and its lack of discrimination between substrate proteins thereby preventing toxicity. IPTG was introduced to induce Abl protein expression in both WT and mutant cultures. A Bradford assay was used to quantify the concentrations of WT and mutant proteins that were obtained through cell lysis, Ni-NTA affinity chromatography, and dialysis; Figures 5 and 6 show the WT and mutant concentrations respectively. SDS-PAGE gel was used to check the purity of both WT and mutant Abl. Figure 7 depicts the pure WT protein with attached hexahistidine tag in lane 9 with a band between 37kDa and 25kDa which is where the Abl protein should be with a size of 32kDa. There is also a band between 75kDa and 50kDa. YopH protein, according to the protein data base, is just over 70kDa which if bound to hexahistidine could have made it in to the pure protein.

There were more impurities observed in the SDS-PAGE of the mutant protein as seen in figure 8. The three bands found in the WT gel were also seen in the mutant gel including the Abl protein along with two others. These proteins could have bound to the column and been eluted along with the protein of interest. If there was error in any of the buffers used during each step of the purification process, other proteins could have changed in a way that allowed them to bind to the column. A kinase assay was used to test the WT and mutant Abl kinase domains in the presence or absence of Gleevec and the results can be seen in figure 9. In both the WT and mutant samples the specific activity was hindered when Gleevec was introduced which was expected due to its competition with ATP in the hinge region where the binding pocket is located as seen in both figures 12 and 13. In Gleevec, only the pyridine and pyrimidine rings interfere directly with the ATP binding site by blocking the binding of the adenine base of ATP. The rings are stabilized inside the binding site by hydrogen binding, van der Walls forces, and hydrophobic interactions. There are hydrogen bonds at Met318, Thr315, Phe311, Asn322. The S417Y mutation in the inactive site of the kinase increased the specific activity. It was hypothesized that the mutation would destabilize the inactive conformation therefore allowing ATP to bind more readily. In the inactive conformation the DFG motif is flipped 180 degrees, Asp381

can no longer interact with Mg-ATP, and Phenylalanine points into to binding site; all opposing the binding of ATP. The mutation opposes this confirmation, therefore allowing a higher specific activity. Although the results confirm our hypothesis, it was with little significance (9.3% increase in specific activity in mutant compared to WT). To improve upon this experiment, better purification should be performed to make sure the protein of interest is in high concentration. Because the protein tested was not pure, the Bradford assay was inaccurate (detects any protein) so the concentration of our target protein could not be accurately determined. This could explain the low specific activity observed in the mutant protein.

Conclusion

The purpose of this experiment was to understand how CML patients can become resistant to Gleevec treatment through mutations of Abl kinase. This was confirmed through experimentation, specifically the S417Y mutation. Further experiments should consider other mutations common in CML patients that could possibly affect the binding of Gleevec. From these mutations new drugs could be developed to fit the geometry and binding properties required.

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