Literature DB >> 35511790

Fast in-vitro screening of FLT3-ITD inhibitors using silkworm-baculovirus protein expression system.

Naoki Yamamoto¹, Jiro Kikuchi², Yusuke Furukawa², Naoya Shibayama¹.

Abstract

We report expression and purification of a FLT3 protein with ITD mutation (FLT3-ITD) with a steady tyrosine kinase activity using a silkworm-baculovirus system, and its application as a fast screening system of tyrosine kinase inhibitors. The FLT3-ITD protein was expressed in Bombyx mori L. pupae infected by gene-modified nucleopolyhedrovirus, and was purified as an active state. We performed an inhibition assay using 17 kinase inhibitors, and succeeded in screening two inhibitors for FLT3-ITD. The result has paved the way for screening FLT3-ITD inhibitors in a fast and easy manner, and also for structural studies.

Entities: Chemical

Mesh：

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Year: 2022 PMID： 35511790 PMCID： PMC9070948 DOI： 10.1371/journal.pone.0261699

Source DB: PubMed Journal: PLoS One ISSN： 1932-6203 Impact factor: 3.240

1. Introduction

Acute myeloid leukemia (AML) is a proliferative malignancy of hematopoietic cells which are blocked in differentiation at a variety of stages [1]. Several mutations in proteins, such as tyrosine kinases, transcription factors, and epigenetic factors, are related to the onset of AML [2]. Among them, FLT3 (Fms-Like Tyrosine kinase-3) with internal tandem duplication (ITD) occurring at the juxtamembrane (JM) domain (Fig 1A, gray and yellow parts), which we term FLT3-ITD, is known to be a poor-prognostic factor [3,4]. The mutation results in constitutive activation of FLT3 tyrosine kinase due to ligand-independent homodimerization. Several drugs have been known to be effective to suppress the activity of FLT3-ITD [5,6]. For example, midostaurin (PKC412) is one of the potent drugs, and has clinically been used [7]. Recently, gilteritinib (ASP2215) has also been released to the market [8]. Yet discovery of other potent drugs is desired for overcoming resistance to existing drugs, and for broadening choices of the clinical treatment.

Fig 1

(A) The duplicated and template (indicated by yellow) amino acid sequence of the FLT3-ITD protein. Bottom is the crystal structure of the WT FLT3 protein in complex with giliteritinib (PDB ID; 6JQR). The gray, red, green, and blue parts represent the JM domain, N robe, activation loop, and C robe, respectively. The yellow part within the JM domain is the template sequence for the duplication. (B) SDS-PAGE of the purification process. Lane 1 is the supernatant of the ground pupae collected by ultracentrifuge. Lane 2 represents the purified protein by the ion-exchange chromatography, and the result of the western blot using anti-FLAG antibody is shown Lane 3. (C, top) Time dependent measurement of the tyrosine kinase activity of the purified FLT3-ITD protein. As a control, midostaurin was used to inhibit the activity. (C, bottom) The inhibitory curves of midostaurin and gilteritinib. The minimal data sets used for these figures are represented in Supporting Information. Drug screening for AML has been performed using cell-proliferation systems in which AML is stimulated upon mutation on related genes. The effect of drugs can be confirmed from the death of the cells. However, the span for cell cultivation required to confirm the effect of drugs usually takes more than two days [7,9-11], and also there still remains a possibility that the regression of the cell life is not directly caused by the suppression of the protein function stimulated by the mutation [12]. Therefore, direct evaluation of the effect of drugs on the target protein is desirable for the more rapid and accurate drug screening. To date, there have been some commercially-available FLT3-ITD proteins by which the kinase activity can be evaluated [13]. However, since the purity of these proteins is not sufficiently high, well-purified FLT3-ITD proteins are desired for biochemical and especially for structural studies. In this study, we report a construction of an expression system for a FLT3-ITD protein using silkworm pupae, and its adaptation to high-throughput drug screening system. The protein was able to be purified as a single component from the pupae as an active state. A drug screening using several candidates was performed, and potent drugs to FLT3-ITD were successfully identified. As far as we know, it is the first time to obtain active recombinant FLT3-ITD proteins as a single component.

2. Materials and methods

2.1. Recombinant baculovirus production for the expression of the FLT3-ITD protein

The FLT3-ITD protein was expressed in the Silkworm-Baculovirus System produced by ProCube (Sysmex Corporation, Kobe, Japan). Briefly, a gene sequence corresponding to the tyrosine kinase domain with an ITD mutation (Fig 1A) was amplified via PCR using primers (forward: 5’-cgcggtaccATGCACAAGTACAAAAAGCAATTTA and reverse: 5’-ggctctagaCACATTCTGATACATCGCTTCTTCTG where the lower-case characters indicate codons for the further ligation reactions and the capital ones part of the FLT3-ITD gene). The amplified oligo DNA was inserted in the pM23 transfer vector (Sysmex Corporation, Kobe, Japan) at KpnI and XbaI cites. The pM23 vector is designed to conjugate the FLAG tag sequence to the C-terminal of the recombinant protein. This gene-transferred pM23 vector was co-transfected with linearized B. mori nucleopolyhedrovirus (i.e. BmNPV) DNA [14] into a B. mori-derived cell line (i.e. BmN) [15]. If the homologous recombination between these two DNA successfully occurs, the recombinant virus obtains the replication function. After 7 days of incubation at 25°C, the recombinant virus existing in the supernatant was injected into the body of silkworm pupae. 50 μl of the virus at 105∼106 PFU/ml was used for the infection. The infected pupae were harvested for 6 days at 25 ºC and 50% humidity, and frozen.

2.2. Purification of the FLT3-ITD protein

The pupae infected by the baculovirus were homogenized with a buffer solution (20 mM Tris-HCl, 150 mM NaCl, 5 mM DTT, 1mg/ml phenylthiourea, one inhibitor cocktail tablet (EASYpack, Roche, Basel, Switzerland) per 50 ml solution, pH 8.0). The homogenized solution was centrifuged to separate the supernatant and precipitate using an ultracentrifuge (100,000 g) for 1 h at 4°C. The supernatant was filtered using 0.8 μm Minisart Syringe Filter (Sartorius, Göttingen, Germany), and purified using DDDDK-tagged protein purification gel (MBL, Japan) followed by elution with 0.1 mg/ml FLAG Peptide (Sigma-Aldrich Co., St. Louis, Missouri, the US). To remove the flag peptide, further purification by the ion-exchange chromatography was performed using ToyoScreen SuperQ-650M column (TOSOH, Yamaguchi, Japan) built up with AKTA purifier (GE healthcare, Chicago, Illinois, the US). The purified FLT3-ITD protein was used for the kinase activity measurements. The concentrations of the protein used for the kinase activity measurement were 0.2–0.4 μM which were estimated by Western Blot using an anti-FLAG antibody, Anti-DDDDK-tag mAb-HRP-Direct (MBL, Japan).

2.3. Kinase activity measurement

The kinase activity of the FLT3-ITD protein was measured by using ADP-Glo™ Kinase Assay Kit (Promega, Madison, Wisconsin, the US). Briefly, the protein solution was incubated in 0.2 mM DTT, 0.1 mg/ml myelin basic protein, 0.1 mM ATP, the reaction buffer, and 10% DMSO. The drug concentration at 1 μM was used for monitoring the inhibition activities. The kinase reaction was initiated upon the addition of ATP, and 15 μl of the reaction solution was mixed with the same amount of ADP-Glo Solution. 40 min after the incubation, 30 μl of Kinase Detection Reagent was added to the solution. 30 min after the incubation, the luminescence intensity was monitored using SPARK 10M (TECAN, Zurich, Switterland) in a 96-wel plate. The measurement was replicated three times and the standard deviation at 95% confidence interval was calculated. To draw the inhibitory curves, the protein solution in the presence of inhibitors at various concentrations was incubated for 2 h and the same detection protocol was performed.

3. Results and discussion

3.1. Purification of the FLT3-ITD protein

In this study, we chose a FLT3-ITD protein found in a clinical case (Fig 1A) [16]. The full amino-acid sequence expressed in this study is shown in Supporting Information. PQYFYVDF is the duplicated sequence where NEYFYVDF prior to the sequence was supposed to be the template of the duplication (indicated by the yellow highlight). The purification by the FLAG tag already yielded an almost pure protein solution. The ion-exchange chromatography was performed to exclude the FLAG peptide, and it was confirmed that the purified FLT3-ITD protein appeared as a single band on SDS-PAGE stained with Coomassie brilliant blue (Fig 1B). The proper expression of the protein was also confirmed by Western Blot using anti-FLAG antibody (Fig 1B). The protein yield was ~20 μg per one pupa.

3.2. The kinase activity of the expressed FLT3-ITD protein

Time-dependence of the kinase activity of the FLT3-ITD protein is shown in Fig 1C top. The kinase activity increases in a time-dependent manner. On the contrary, no such increase was observed in the presence of the inhibitor, midostaurin. The results represent that the expressed protein possesses a steady kinase activity, which is detectable by the standard assay method. The absolute protein activity was calculated using a standard calibration method, and the ATP-to-ADP conversion rate was obtained to be ~50 nmol/min/mg. The inhibition curves by midostaurin and gilteritinib are shown in Fig 1C bottom. These two inhibitors certainly inhibited the kinase activity in the concentration-dependent manner. The inhibition curves were fitted by the hill function to obtain the IC50 values. As a result, the IC50 values were 4.2±0.1 nM and 16±3 nM (S.D. obtained by the curve fitting) for midostaurin and gilteritinib, respectively. The value of midostaurin is two-order lower than that reported in in-vivo study [7], indicating that our screening system possesses a high sensitivity.

3.3. Screening assay of various potentially-useful drugs against the expressed FLT3-ITD protein

The inhibition activity of various kinds of kinase inhibitors was monitored. We tried 17 kinase inhibitors that have already known to inhibit kinase activities. Fig 2A shows the inhibitory activities of the compounds. As controls, midostaurin (positive control) and buffer (negative control) were also tested. Sorafenib and sunitinib, which have been known to be effective to FLT3-ITD mutated cell lines [17], had clear inhibitory effects whereas the other drugs did not show apparent activities. The inhibitory curves of the two drugs were obtained and fitted by using the hill function as shown in Fig 2B. As a result, the IC50 values were 4.6±0.9 nM and 8.2±4.3 nM (S.D. obtained by curve fitting) for sorafenib and sunitinib, respectively. These values are as low as those of midostaurin, confirming that these two drugs are potent inhibitors of the FLT3-ITD protein.

Fig 2

(A) Inhibition of the tyrosine kinase activity of the FLT3-ITD protein by various drugs. Sorafenib and sunitinib possess the same activity as that of midostaurin. (B) The inhibitory curves of sorafenib and sunitinib. The minimal data sets used for these figures are represented in Supporting Information.

4. Conclusion

As far as we know, it is the first time to successfully express and purify soluble FLT3-ITD protein. Thus, the present expression system using the silkworm pupae is a quite effective way to obtain the active FLT3-ITD protein. In the screening experiment, sorafenib and sunitinib were successfully found to be effective to inhibit the tyrosine kinase activity of the FLT3-ITD protein. The screening is fast (within 2 hours) and ~1,000 trials can be done per one pupa. Therefore, the expression system built in this study is a powerful method to find new tyrosine kinase inhibitors. Furthermore, the protein obtained as a highly-purified state is suitable for biochemical and especially for structural determination studies. (DOCX) Click here for additional data file. (PDF) Click here for additional data file. 16 Feb 2022

PONE-D-21-38649

Fast in-vitro screening of FLT3-ITD inhibitors using silkworm-baculovirus protein expression system

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Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here. Reviewer #1: Yes Reviewer #2: Yes ********** 5. Review Comments to the Author Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #1: In the era of personalized medicine, it becomes important to have effective and fast techniques for the detection of active molecules. This work clearly and rigorously presents a useful and fast system for this type of activity. The point that could be developed in the future is the study of the different lengths of the ITD on the efficacy of the inhibition. Reviewer #2: Major comments： 1. To date, there have been some commercially-available FLT3-ITD proteins by which the kinase activity can be evaluated. Why are you still doing this work? please provide evidence if you purified protein better than the commercial one. 2. In the abstract， “We performed an inhibition assay using 17 potential kinase inhibitors, and succeeded in identifying two potent inhibitors for FLT3-ITD.” It is easy to mislead readers into thinking that you have found two new inhibitors. Minor comments： Some details of the materials and methods need to be supplemented, the following are specific examples: 1. What is the template for ITD mutation sequence PCR? 2. When co-transfected pM23 vector with linearized BmNPV DNA, what is the transfection reagent and what is the amount of transfection system? And what are the cell culture conditions? 3. Obviously, not all of the homologous recombination between the two DNA can successfully occurs, how did you screen that? 4. What is amount of the recombinant virus used for injection? And after injection, what conditions are the pupae placed in? Next are some of my questions or suggestions: 1. During the 7 days incubation period after transfection, I would like to know if you change the medium for the old cells or collect the recombinant virus to re-infect the new cells? 2. Fig 1. (B) Lane 1 and Lane 2 appear to be two images stitched together; and I think adding one lane, the supernatant of the normal pupae (blank control) would be better. 3. In the Results and discussion, 3.1, SDS PAGE missed a “-”. 4. In conclusion, “Thus, the present expression system using the silkworm pupae is quite effective way to obtain the active FLT3-ITD protein”, before “quite” need to add “a”. 5. The references may be a little less, and mostly not up to date. ********** 6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files. If you choose “no”, your identity will remain anonymous but your review may still be made public. Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy. Reviewer #1: No Reviewer #2: No [NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.] While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step. 3 Mar 2022 Response to the reviewers’ comments (The yellow highlighted parts are those modified in the main text) Reviewer #1: In the era of personalized medicine, it becomes important to have effective and fast techniques for the detection of active molecules. This work clearly and rigorously presents a useful and fast system for this type of activity. The point that could be developed in the future is the study of the different lengths of the ITD on the efficacy of the inhibition. Reply We appreciate that the importance of our study is well understood. As the reviewer mentioned, we would like to deal with FLT-ITD proteins with different ITD lengths, so that new drugs optimized for the personalized medicine will be efficiently found. Reviewer #2: Major comments： 1. To date, there have been some commercially-available FLT3-ITD proteins by which the kinase activity can be evaluated. Why are you still doing this work? please provide evidence if you purified protein better than the commercial one. Reply Thank you for giving a comment discussing about the importance of our study. We would like to answer them by the point-by-point manner. As the reviewer mentioned, there has already been commercially-available FLT3-ITDs. For example, SignalChem supplies a FLT3-ITD protein (https://signalchem.com/product_details.php?id=ZWE1ZDJmMWM0NjA4MjMyZTA3ZDNhYTNkOTk4ZTUxMzUtMTE5MDM=#specifications). However, the purity of the protein is low because it contains non-negligible contaminated proteins around 26 kDa (Figure R1). On the other hand, in our case the FLT3-ITD protein appears as the major band in the SDS-PAGE as shown in Figure 1B, which represents that we purified protein better than the commercial case. 2. In the abstract， “We performed an inhibition assay using 17 potential kinase inhibitors, and succeeded in identifying two potent inhibitors for FLT3-ITD.” It is easy to mislead readers into thinking that you have found two new inhibitors. Reply Following what the reviewer suggested, we have changed the corresponding sentence as follows; (Page 2, line 6) We performed an inhibition assay using 17 kinase inhibitors, and succeeded in screening two inhibitors for FLT3-ITD. In addition to this, one sentence in conclusion was also modified as follows; (page 10, line 3) As far as we know, it is the first time to successfully express and purify soluble FLT3-ITD protein. (Original sentence; As far as we know, it is the first time to successfully express a soluble FLT3-ITD protein.) Minor comments： Some details of the materials and methods need to be supplemented, the following are specific examples: 1. What is the template for ITD mutation sequence PCR? Reply We added the DNA sequence confirmed in the pM23 vector in Supporting Information. 2. When co-transfected pM23 vector with linearized BmNPV DNA, what is the transfection reagent and what is the amount of transfection system? And what are the cell culture conditions? Reply The transfection reagent and its amount are not open because such information is protected under the patent of ProCube system owned by Sysmex Corporation. The cell culture was harvested at 25 ºC. We added this information in Materials and Methods. 3. Obviously, not all of the homologous recombination between the two DNA can successfully occurs, how did you screen that? We did not confirm if each cell was infected. However, we were able to tell that 80-90 % of the cells were infected by the virus judging from their appearance. 4. What is amount of the recombinant virus used for injection? And after injection, what conditions are the pupae placed in? Reply 50 μl of the virus at 105～106 PFU/ml was used for the infection. The infected pupae were put at 25 ºC and 50 % humidity. We added these experimental conditions in Materials and Methods. Next are some of my questions or suggestions: 1. During the 7 days incubation period after transfection, I would like to know if you change the medium for the old cells or collect the recombinant virus to re-infect the new cells? Reply We did not change the medium. 2. Fig 1. (B) Lane 1 and Lane 2 appear to be two images stitched together; and I think adding one lane, the supernatant of the normal pupae (blank control) would be better. Reply Since we do not find the merit to add the supernatant of the normal pupae to the SDS-PAGE result, we do not include it. 3. In the Results and discussion, 3.1, SDS PAGE missed a “-”. Reply We added the missing hyphen at the corresponding place. 4. In conclusion, “Thus, the present expression system using the silkworm pupae is quite effective way to obtain the active FLT3-ITD protein”, before “quite” need to add “a”. Reply We added the missing word at the corresponding place. 5. The references may be a little less, and mostly not up to date. Reply We added some up-to-date references in Introduction. Submitted filename: Response_to_reviewers_PlosOne_220222.docx Click here for additional data file. 30 Mar 2022 Fast in-vitro screening of FLT3-ITD inhibitors using silkworm-baculovirus protein expression system PONE-D-21-38649R1 Dear Dr. Yamamoto, Sorry for the delay. One of the two original reviewers was no longer available so the process was longer than usual as I had to check that all her/his requests were fullfilled. 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For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified. Reviewer #1: Yes ********** 5. Is the manuscript presented in an intelligible fashion and written in standard English? PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here. Reviewer #1: Yes ********** 6. Review Comments to the Author Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. 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Your manuscript is now with our production department. If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org. If we can help with anything else, please email us at plosone@plos.org. Thank you for submitting your work to PLOS ONE and supporting open access. Kind regards, PLOS ONE Editorial Office Staff on behalf of Dr. Francesco Bertolini Academic Editor PLOS ONE

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Authors: D Bonnet; J E Dick
Journal: Nat Med Date: 1997-07 Impact factor: 53.440

3. Efficient protein production using a Bombyx mori nuclear polyhedrosis virus lacking the cysteine proteinase gene.

Authors: T Suzuki; T Kanaya; H Okazaki; K Ogawa; A Usami; H Watanabe; K Kadono-Okuda; M Yamakawa; H Sato; H Mori; S Takahashi; K Oda
Journal: J Gen Virol Date: 1997-12 Impact factor: 3.891

Review 4. The molecular mechanisms behind activation of FLT3 in acute myeloid leukemia and resistance to therapy by selective inhibitors.

Authors: Ran Friedman
Journal: Biochim Biophys Acta Rev Cancer Date: 2021-12-08 Impact factor: 11.414

5. Sensitivity toward sorafenib and sunitinib varies between different activating and drug-resistant FLT3-ITD mutations.

Authors: Rama Krishna Kancha; Rebekka Grundler; Christian Peschel; Justus Duyster
Journal: Exp Hematol Date: 2007-10 Impact factor: 3.084

6. Structural and numerical variation of FLT3/ITD in pediatric AML.

Authors: Soheil Meshinchi; Derek L Stirewalt; Todd A Alonzo; Titus J Boggon; Robert B Gerbing; Jennifer L Rocnik; Beverly J Lange; D Gary Gilliland; Jerald P Radich
Journal: Blood Date: 2008-02-27 Impact factor: 22.113

7. Spectrum and prognostic relevance of driver gene mutations in acute myeloid leukemia.

Authors: Klaus H Metzeler; Tobias Herold; Maja Rothenberg-Thurley; Susanne Amler; Maria C Sauerland; Dennis Görlich; Stephanie Schneider; Nikola P Konstandin; Annika Dufour; Kathrin Bräundl; Bianka Ksienzyk; Evelyn Zellmeier; Luise Hartmann; Philipp A Greif; Michael Fiegl; Marion Subklewe; Stefan K Bohlander; Utz Krug; Andreas Faldum; Wolfgang E Berdel; Bernhard Wörmann; Thomas Büchner; Wolfgang Hiddemann; Jan Braess; Karsten Spiekermann
Journal: Blood Date: 2016-06-10 Impact factor: 22.113

8. Activating JAK-mutations confer resistance to FLT3 kinase inhibitors in FLT3-ITD positive AML in vitro and in vivo.

Authors: Christoph Rummelt; Sivahari P Gorantla; Manja Meggendorfer; Anne Charlet; Cornelia Endres; Konstanze Döhner; Florian H Heidel; Thomas Fischer; Torsten Haferlach; Justus Duyster; Nikolas von Bubnoff
Journal: Leukemia Date: 2020-11-04 Impact factor: 11.528

9. The anti-mitotic agents PTC-028 and PTC596 display potent activity in pre-clinical models of multiple myeloma but challenge the role of BMI-1 as an essential tumour gene.

Authors: Arnold Bolomsky; Joséphine Muller; Kathrin Stangelberger; Margaux Lejeune; Elodie Duray; Helene Breid; Louise Vrancken; Christina Pfeiffer; Wolfgang Hübl; Martin Willheim; Marla Weetall; Art Branstrom; Niklas Zojer; Jo Caers; Heinz Ludwig
Journal: Br J Haematol Date: 2020-03-30 Impact factor: 6.998

Review 10. Potential targeting of FLT3 acute myeloid leukemia.

Authors: Alexander J Ambinder; Mark Levis
Journal: Haematologica Date: 2021-03-01 Impact factor: 9.941