- Email: [email protected]
Identification of human lactate dehydrogenase A inhibitors with anti-osteosarcoma activity through cell-based phenotypic screening
Bioorganic & Medicinal Chemistry Letters xxx (xxxx) xxxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters journal homepage: www.elsevier.com/locate/bmcl
Identification of human lactate dehydrogenase A inhibitors with antiosteosarcoma activity through cell-based phenotypic screening☆ Fei Wanga, Qiheng Zhaoa, Junzhi Liub, Zhe Wangc, Daliang Konga,
⁎
a
Department of Orthopaedics, Jilin University China-Japan Union Hospital, Jilin 130030, China Department of Quality Control, Jilin University China-Japan Union Hospital, Jilin 130030, China c Department of Pathology, School of Basic Medicine, Jilin University, Jilin 130000, China b
ARTICLE INFO
ABSTRACT
Keywords: Lactate dehydrogenase A Glycolysis Anticancer Cancer metabolism
Human lactate dehydrogenase A plays a key role in the glycolytic process, the inhibition of the enzyme is therefore considered of interest in developing anticancer therapeutics. However, due to the highly polar nature of hLDHA binding pocket, it is very challenge to discover potent cellular active hLDHA inhibitor. Combined a cell-based phenotypic screening assay with a primary enzymatic assay, we discovered three cellular active hLDHA inhibitors, namely 38, 63, and 374, which reduced MG-63 cell proliferation with IC50 values of 6.47, 2.93, and 6.10 µM, respectively, and inhibited hLDHA with EC50 values of 3.03, 0.63, and 3.26 µM, respectively.
Most of cancer cells feature an elevated of aerobic glycolysis that convert of glucose into lactate in cytoplasm rather than mitochondrial oxidative phosphorylation even in aerobic conditions.1 This altered metabolic pathway is recognized as a hallmark of cancer.2 Disturbing the process is believed to be a promising strategy for cancer treatment.3 Human lactate dehydrogenase A (hLDHA) is a glycolytic enzyme that catalyzes the interconversion of pyruvate to lactate with simultaneous oxidation of NADH to NAD+ in cytoplasm, which is essential to maintain the glycolytic flow.4 This inefficient metabolism could supply cancer cells with rapid energy and enough precursors for proliferation. So the inhibition of hLDHA function to rectify this abnormal metabolic process could be a promising strategy for developing anticancer therapeutics.5 In addition, the overexpression of hLDHA is often observed in various human solid tumors, such as osteosarcoma,6 MiaPaCa-2 cell,7 lung cancer,8 glioma cell,9 which makes hLDHA is a potentially important therapeutic target to mitigate the glycolysis pathway in cancer cells.10 Though hLDHA inhibitors have been sought for over a decade, and these efforts have yielded a large number of potent hLDHA inhibitors. However, most of these inhibitors are lack of cellular activity. For ex-
ample, optimization of high throughput screening hits 1 and 2 (Fig. 1) have been reported,11,12 but the resulted compounds exhibited poor physiochemical properties. Compound 3 (Fig. 1) has been demonstrated to give a low nanomolar biochemical activity on hLDHA,13 however, the compound displayed limited cellular activity, which prevent its further clinical application. Considering the fact that most of the reported hLDHA inhibitors are lack of cellular activity, we herein developed a cell-based phenotypic assay by screening of an in-house small molecule library, from which we identified some candidates with cellular activity, then measured their hLDHA inhibitory potency. With these biological evaluations, we hope to discover some cellular active hLDHA inhibitors for cancer treatment. Firstly, we measured the hLDHA expression level on MG-63, U20S, and HOS osteosarcoma cells and L02 human normal cell, and found that hLDHA is overexpressed in osteosarcoma cells as compared to normal one (Fig. S1 in Supporting Information). We then screened an in-house small molecule library containing 500 commercially available compounds against MG-63 cell proliferation to identify antiproliferative candidates. We fixed compounds screening concentration at 10 µM, and MG-63 cell density was 4000 per well, cisplatin and doxorubicin were
All the experimental details were included in the Supplementary lnformation. Corresponding author. E-mail address: [email protected] (D. Kong).
☆ ⁎
https://doi.org/10.1016/j.bmcl.2019.126909 Received 14 November 2019; Received in revised form 10 December 2019; Accepted 12 December 2019 0960-894X/ © 2019 Elsevier Ltd. All rights reserved.
Please cite this article as: Fei Wang, et al., Bioorganic & Medicinal Chemistry Letters, https://doi.org/10.1016/j.bmcl.2019.126909
Bioorganic & Medicinal Chemistry Letters xxx (xxxx) xxxx
F. Wang, et al.
Fig. 1. Structure of representative hLDHA inhibitors.
used as positive control. As shown in Table S1 (see Supporting Information), we discovered 42 compounds (highlighted as yellow in Table S1). All of the identified compounds (Fig. S2 in Supporting Information) displayed > 50% inhibitory rates against MG-63 cell proliferation. We then conducted a biochemical assay that the 42 compounds were tested by an enzymatic assay using hLDHA in the presence of pyruvate and NADH, then monitor the decrease of absorbance at 340 nm in a microplate reader. As shown in Fig. 2A, most of the compounds exhibited poor LDHA inhibitory rates (< 50%) at 5 μM, we still observed three compounds, namely 38, 63, and 374 (Fig. 2B), which gave promising anti-hLDHA activities (> 50%). Especially, 63 gave the best inhibitory rate of 91.2% at 5 µM. We therefore measured full dose-response manners of the three compounds against hLDHA. As shown in Fig. 2C, the EC50 values were 3.03, 0.67, and 3.26 µM for the three compounds, respectively. Then we titrated 38, 63, and 374 into hLDHA in an isothermal titration calorimetry (ITC) experiment (Fig. 2D), the Kd values were 6.41, 2.41, and 9.85 µM, respectively, which is in line with the biochemical data, indicating the three compounds interacted and inhibited hLDHA. To further verify the three compounds interacted with hLDHA, we docked them into the binding pocket of hLDHA (PDB code: 4ZVV) by using SYBYL-X 2.0 software. The N-oxide group14 in three compounds formed direct hydrogen bond interactions with the amino acids His192 and Asn137, as shown in Fig. 2E. And others groups in the three compounds were observed to form hydrogen bond with Asp168. All the three amino acids played an important biofunctional roles for hLDHA in the catalytic process, suggesting the three compounds bound to hLDHA, another evidence that consist with our in vitro enzymatic assay. The three compounds showed most potent enzymatic activities, therefore we studied their anticancer activities against several osteosarcoma cells. As shown in Fig. 3A, 63 at 5 µM reduced MG-63 cell growth in a time-dependent manner, as indicated from the IncuCyte Zoom Live-Cell analysis. Then we tested IC50 values of the three compounds, and the IC50 values were 6.47, 2.93, and 6.10 µM, respectively, as shown in Fig. 3B. Moreover, they inhibited other osteosarcoma cells including HOS, Saos-2, and U20S, with the IC50 values ranging from 3.7 to 14.9 µM (Fig. 3C). Finally, 63 at 5 µM incubated with MG-63 cell for 12, 18, and 24 h, and the apoptosis cells were detected by Annexin V-
FITC/PI FACS assay in a flow cytometry. As shown in Fig. 3D, the percentages of apoptotic population in MG-63 cells for each time were 5.42, 11.69, and 23.33%, respectively, following a time-dependent manner. Then 63 at 2, 5, 10 µM incubated with MG-63 cell for 24 h (Fig. 3E), the percentages of apoptotic population were 3.61, 13.38, and 23.58%, displaying a dose-dependent manner. Targeting hLDHA should alter the metabolic profile of cancer cell by forming the lactate production rather than mitochondrial oxidative phosphorylation, so the lactate formation, oxygen consumption rate (OCR), extracellular acidification rates (ECAR), and proton production rate (PPR) in cancer cell should change accordingly. The metabolic modulatory effect of 63 was evaluated on a Seahorse XFe24 extracellular flux analyzer. As shown in Fig. 4A, the lactate formation was reduced in MG-63 cells culturing medium after treating with 63 for 4 h, while the lactate formation show little change in human normal cell after treating 63 (Fig. S3 in Supporting Information). Similar to the lactate formation, ECAR (Fig. 4C) and PPR (Fig. 4D) were significantly decreased with the addition of 63. This could be explained by the reduction of glycolysis in the cancer cells after treating of 63. In Fig. 4B, we observed a dose-dependent increase in OCR, suggesting a metabolic alteration caused by 63 treatment. In summary, we herein report the discovery of anti-osteosarcoma therapeutics that target to hLDHA. We screened an in-house small molecule library that contains 500 commercial available compounds against MG-63 cell proliferation. From which, we identified 42 antiosteosarcoma compounds, and evaluated their anti-hLDHA activities. We found that the three compounds showed promising inhibitory activity to hLDHA, and bound to hLDHA with Kd values of 6.41, 2.41, and 9.85 µM, inhibited hLDHA activity with EC50 values of 3.03, 0.63, and 3.26 µM, respectively, and reduced MG-63 cell proliferation with IC50 values of 6.47, 2.93, and 6.10 µM. In addition, 63 significantly modulated pyruvate metabolic profile in MG-63 cells by decreasing ECRA, PPR and lactate formation, and increasing OCR values. All these observations indicated that 63 could be used as a promising lead for the development of potent cellular active hLDHA inhibitor. Further exploitation of the chemical modification of 63 and pharmacology study are ongoing and will be reported in due time.
2
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Fig. 2. Compounds inhibited hLDHA activity. (A) The compounds from cell-based phenotypic screening inhibited hLDHA activity, the concentration of all the compounds was 5 µM. (B) Chemical structures of 38, 63, and 374. (C) EC50 values of 38, 63, and 374 against hLDHA. (D) The Kd values of the three compounds bound to hLDHA, the data were derived from the titration of the three compounds into the hLDHA protein by isothermal titration calorimetry (ITC). (E) Docking study of the three compounds on the binding pocket of hLDHA. The amino acids involved in hydrogen bond interactions were shown as stick and colored as red, yellow dashes represents the hydrogen bonds. The graphics of 3D views were drawn by PyMOL.
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Fig. 3. The three compounds reduced osteosarcoma cell proliferation. (A) Growth of MG-63 cells was monitored by IncuCyte after being treated with 64 at different time; (B) IC50 values of the three compounds against MG-63 cell; (C) Anti-osteosarcoma activity of the three compounds on different osteosarcoma cell; (D) Timedependent manner of 63 caused MG-63 cell apoptosis; (E) Dose-dependent manner of 63 caused MG-63 cell apoptosis. MG-63 cell was treated with 63, then stained with annexin V/PI.
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Fig. 4. Bioenergetics changes of MG-63 cells after treating with 63. (A) Compound 63 at doses of 2, 5, and 10 µM reduced MG-63 lactate formation; (B–D) MG-63 cells were treated with 63 for 4 h, then OCR/ECAR/PPR were measured. *P < 0.05, versus the control group.
Declaration of Competing Interest
2013;12:829; (b) Warburg O. Science. 1956;123:309. 2. Tennant DA, Durán RV, Gottlieb E. Nat Rev Cancer. 2010;10:267. 3. (a) Cairns RA, Harris IS, Mak TW. Nat Rev Cancer. 2011;11:85;
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
(b) Vander Heiden MG, Cantley LC, Thompson CB. Science. 2009;324:1029. 4. (a) Augoff K, Hryniewicz-Jankowska A, Tabola R. Cancer Lett. 2015;358:1;
Acknowledgement
(b) Zhang SL, He Y, Tam KY. Drug Discov Today. 2018;23:1407;
This work was supported by special fund for key laboratory of science and technology department of Jilin province (20190201282JC).
(c) Rani R, Kumar V. J Med Chem. 2016;59:487. 5. (a) Purkey HE, Robarge K, Chen J, et al. ACS Med Chem Lett. 2016;7:896;
Appendix A. Supplementary data
6. 7. 8. 9. 10. 11. 12. 13. 14.
Supplementary data to this article can be found online at https:// doi.org/10.1016/j.bmcl.2019.126909. References 1. (a) Galluzzi L, Kepp O, Vander Heiden MG, Kroemer G. Nat Rev Drug Discov.
5
(b) Xie H, Hanai J-I, Ren J-G, et al. Cell Metab. 2014;19:795. Cui W, Lv W, Qu Y, et al. Bioorg Med Chem Lett. 2016;26:3984. Boudreau A, Purkey HE, Hitz A, et al. Nat Chem Biol. 2016;12:779. Hou XM, Yuan SQ, Zhao D, Liu XJ, Wu XA. Biosci Rep. 2019;39 pii: BSR20181476. Di H, Zhang X, Guo Y, et al. Oncol Lett. 2018;15:5131. Rai G, Brimacombe KR, Mott BT, et al. J Med Chem. 2017;60:9184. Dragovich PS, Fauber BP, Corson LB, et al. Bioorg Med Chem Lett. 2013;23:3186. Fauber BP, Dragovich PS, Chen J, et al. Bioorg Med Chem Lett. 2013;23:5533. Ward RA, Brassington C, Breeze AL, et al. J Med Chem. 2012;55:3285. Lumeras W, Vidal L, Vidal B, et al. J Med Chem. 2011;54:7899.
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters journal homepage: www.elsevier.com/locate/bmcl
Identification of human lactate dehydrogenase A inhibitors with antiosteosarcoma activity through cell-based phenotypic screening☆ Fei Wanga, Qiheng Zhaoa, Junzhi Liub, Zhe Wangc, Daliang Konga,
⁎
a
Department of Orthopaedics, Jilin University China-Japan Union Hospital, Jilin 130030, China Department of Quality Control, Jilin University China-Japan Union Hospital, Jilin 130030, China c Department of Pathology, School of Basic Medicine, Jilin University, Jilin 130000, China b
ARTICLE INFO
ABSTRACT
Keywords: Lactate dehydrogenase A Glycolysis Anticancer Cancer metabolism
Human lactate dehydrogenase A plays a key role in the glycolytic process, the inhibition of the enzyme is therefore considered of interest in developing anticancer therapeutics. However, due to the highly polar nature of hLDHA binding pocket, it is very challenge to discover potent cellular active hLDHA inhibitor. Combined a cell-based phenotypic screening assay with a primary enzymatic assay, we discovered three cellular active hLDHA inhibitors, namely 38, 63, and 374, which reduced MG-63 cell proliferation with IC50 values of 6.47, 2.93, and 6.10 µM, respectively, and inhibited hLDHA with EC50 values of 3.03, 0.63, and 3.26 µM, respectively.
Most of cancer cells feature an elevated of aerobic glycolysis that convert of glucose into lactate in cytoplasm rather than mitochondrial oxidative phosphorylation even in aerobic conditions.1 This altered metabolic pathway is recognized as a hallmark of cancer.2 Disturbing the process is believed to be a promising strategy for cancer treatment.3 Human lactate dehydrogenase A (hLDHA) is a glycolytic enzyme that catalyzes the interconversion of pyruvate to lactate with simultaneous oxidation of NADH to NAD+ in cytoplasm, which is essential to maintain the glycolytic flow.4 This inefficient metabolism could supply cancer cells with rapid energy and enough precursors for proliferation. So the inhibition of hLDHA function to rectify this abnormal metabolic process could be a promising strategy for developing anticancer therapeutics.5 In addition, the overexpression of hLDHA is often observed in various human solid tumors, such as osteosarcoma,6 MiaPaCa-2 cell,7 lung cancer,8 glioma cell,9 which makes hLDHA is a potentially important therapeutic target to mitigate the glycolysis pathway in cancer cells.10 Though hLDHA inhibitors have been sought for over a decade, and these efforts have yielded a large number of potent hLDHA inhibitors. However, most of these inhibitors are lack of cellular activity. For ex-
ample, optimization of high throughput screening hits 1 and 2 (Fig. 1) have been reported,11,12 but the resulted compounds exhibited poor physiochemical properties. Compound 3 (Fig. 1) has been demonstrated to give a low nanomolar biochemical activity on hLDHA,13 however, the compound displayed limited cellular activity, which prevent its further clinical application. Considering the fact that most of the reported hLDHA inhibitors are lack of cellular activity, we herein developed a cell-based phenotypic assay by screening of an in-house small molecule library, from which we identified some candidates with cellular activity, then measured their hLDHA inhibitory potency. With these biological evaluations, we hope to discover some cellular active hLDHA inhibitors for cancer treatment. Firstly, we measured the hLDHA expression level on MG-63, U20S, and HOS osteosarcoma cells and L02 human normal cell, and found that hLDHA is overexpressed in osteosarcoma cells as compared to normal one (Fig. S1 in Supporting Information). We then screened an in-house small molecule library containing 500 commercially available compounds against MG-63 cell proliferation to identify antiproliferative candidates. We fixed compounds screening concentration at 10 µM, and MG-63 cell density was 4000 per well, cisplatin and doxorubicin were
All the experimental details were included in the Supplementary lnformation. Corresponding author. E-mail address: [email protected] (D. Kong).
☆ ⁎
https://doi.org/10.1016/j.bmcl.2019.126909 Received 14 November 2019; Received in revised form 10 December 2019; Accepted 12 December 2019 0960-894X/ © 2019 Elsevier Ltd. All rights reserved.
Please cite this article as: Fei Wang, et al., Bioorganic & Medicinal Chemistry Letters, https://doi.org/10.1016/j.bmcl.2019.126909
Bioorganic & Medicinal Chemistry Letters xxx (xxxx) xxxx
F. Wang, et al.
Fig. 1. Structure of representative hLDHA inhibitors.
used as positive control. As shown in Table S1 (see Supporting Information), we discovered 42 compounds (highlighted as yellow in Table S1). All of the identified compounds (Fig. S2 in Supporting Information) displayed > 50% inhibitory rates against MG-63 cell proliferation. We then conducted a biochemical assay that the 42 compounds were tested by an enzymatic assay using hLDHA in the presence of pyruvate and NADH, then monitor the decrease of absorbance at 340 nm in a microplate reader. As shown in Fig. 2A, most of the compounds exhibited poor LDHA inhibitory rates (< 50%) at 5 μM, we still observed three compounds, namely 38, 63, and 374 (Fig. 2B), which gave promising anti-hLDHA activities (> 50%). Especially, 63 gave the best inhibitory rate of 91.2% at 5 µM. We therefore measured full dose-response manners of the three compounds against hLDHA. As shown in Fig. 2C, the EC50 values were 3.03, 0.67, and 3.26 µM for the three compounds, respectively. Then we titrated 38, 63, and 374 into hLDHA in an isothermal titration calorimetry (ITC) experiment (Fig. 2D), the Kd values were 6.41, 2.41, and 9.85 µM, respectively, which is in line with the biochemical data, indicating the three compounds interacted and inhibited hLDHA. To further verify the three compounds interacted with hLDHA, we docked them into the binding pocket of hLDHA (PDB code: 4ZVV) by using SYBYL-X 2.0 software. The N-oxide group14 in three compounds formed direct hydrogen bond interactions with the amino acids His192 and Asn137, as shown in Fig. 2E. And others groups in the three compounds were observed to form hydrogen bond with Asp168. All the three amino acids played an important biofunctional roles for hLDHA in the catalytic process, suggesting the three compounds bound to hLDHA, another evidence that consist with our in vitro enzymatic assay. The three compounds showed most potent enzymatic activities, therefore we studied their anticancer activities against several osteosarcoma cells. As shown in Fig. 3A, 63 at 5 µM reduced MG-63 cell growth in a time-dependent manner, as indicated from the IncuCyte Zoom Live-Cell analysis. Then we tested IC50 values of the three compounds, and the IC50 values were 6.47, 2.93, and 6.10 µM, respectively, as shown in Fig. 3B. Moreover, they inhibited other osteosarcoma cells including HOS, Saos-2, and U20S, with the IC50 values ranging from 3.7 to 14.9 µM (Fig. 3C). Finally, 63 at 5 µM incubated with MG-63 cell for 12, 18, and 24 h, and the apoptosis cells were detected by Annexin V-
FITC/PI FACS assay in a flow cytometry. As shown in Fig. 3D, the percentages of apoptotic population in MG-63 cells for each time were 5.42, 11.69, and 23.33%, respectively, following a time-dependent manner. Then 63 at 2, 5, 10 µM incubated with MG-63 cell for 24 h (Fig. 3E), the percentages of apoptotic population were 3.61, 13.38, and 23.58%, displaying a dose-dependent manner. Targeting hLDHA should alter the metabolic profile of cancer cell by forming the lactate production rather than mitochondrial oxidative phosphorylation, so the lactate formation, oxygen consumption rate (OCR), extracellular acidification rates (ECAR), and proton production rate (PPR) in cancer cell should change accordingly. The metabolic modulatory effect of 63 was evaluated on a Seahorse XFe24 extracellular flux analyzer. As shown in Fig. 4A, the lactate formation was reduced in MG-63 cells culturing medium after treating with 63 for 4 h, while the lactate formation show little change in human normal cell after treating 63 (Fig. S3 in Supporting Information). Similar to the lactate formation, ECAR (Fig. 4C) and PPR (Fig. 4D) were significantly decreased with the addition of 63. This could be explained by the reduction of glycolysis in the cancer cells after treating of 63. In Fig. 4B, we observed a dose-dependent increase in OCR, suggesting a metabolic alteration caused by 63 treatment. In summary, we herein report the discovery of anti-osteosarcoma therapeutics that target to hLDHA. We screened an in-house small molecule library that contains 500 commercial available compounds against MG-63 cell proliferation. From which, we identified 42 antiosteosarcoma compounds, and evaluated their anti-hLDHA activities. We found that the three compounds showed promising inhibitory activity to hLDHA, and bound to hLDHA with Kd values of 6.41, 2.41, and 9.85 µM, inhibited hLDHA activity with EC50 values of 3.03, 0.63, and 3.26 µM, respectively, and reduced MG-63 cell proliferation with IC50 values of 6.47, 2.93, and 6.10 µM. In addition, 63 significantly modulated pyruvate metabolic profile in MG-63 cells by decreasing ECRA, PPR and lactate formation, and increasing OCR values. All these observations indicated that 63 could be used as a promising lead for the development of potent cellular active hLDHA inhibitor. Further exploitation of the chemical modification of 63 and pharmacology study are ongoing and will be reported in due time.
2
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Fig. 2. Compounds inhibited hLDHA activity. (A) The compounds from cell-based phenotypic screening inhibited hLDHA activity, the concentration of all the compounds was 5 µM. (B) Chemical structures of 38, 63, and 374. (C) EC50 values of 38, 63, and 374 against hLDHA. (D) The Kd values of the three compounds bound to hLDHA, the data were derived from the titration of the three compounds into the hLDHA protein by isothermal titration calorimetry (ITC). (E) Docking study of the three compounds on the binding pocket of hLDHA. The amino acids involved in hydrogen bond interactions were shown as stick and colored as red, yellow dashes represents the hydrogen bonds. The graphics of 3D views were drawn by PyMOL.
3
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Fig. 3. The three compounds reduced osteosarcoma cell proliferation. (A) Growth of MG-63 cells was monitored by IncuCyte after being treated with 64 at different time; (B) IC50 values of the three compounds against MG-63 cell; (C) Anti-osteosarcoma activity of the three compounds on different osteosarcoma cell; (D) Timedependent manner of 63 caused MG-63 cell apoptosis; (E) Dose-dependent manner of 63 caused MG-63 cell apoptosis. MG-63 cell was treated with 63, then stained with annexin V/PI.
4
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Fig. 4. Bioenergetics changes of MG-63 cells after treating with 63. (A) Compound 63 at doses of 2, 5, and 10 µM reduced MG-63 lactate formation; (B–D) MG-63 cells were treated with 63 for 4 h, then OCR/ECAR/PPR were measured. *P < 0.05, versus the control group.
Declaration of Competing Interest
2013;12:829; (b) Warburg O. Science. 1956;123:309. 2. Tennant DA, Durán RV, Gottlieb E. Nat Rev Cancer. 2010;10:267. 3. (a) Cairns RA, Harris IS, Mak TW. Nat Rev Cancer. 2011;11:85;
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
(b) Vander Heiden MG, Cantley LC, Thompson CB. Science. 2009;324:1029. 4. (a) Augoff K, Hryniewicz-Jankowska A, Tabola R. Cancer Lett. 2015;358:1;
Acknowledgement
(b) Zhang SL, He Y, Tam KY. Drug Discov Today. 2018;23:1407;
This work was supported by special fund for key laboratory of science and technology department of Jilin province (20190201282JC).
(c) Rani R, Kumar V. J Med Chem. 2016;59:487. 5. (a) Purkey HE, Robarge K, Chen J, et al. ACS Med Chem Lett. 2016;7:896;
Appendix A. Supplementary data
6. 7. 8. 9. 10. 11. 12. 13. 14.
Supplementary data to this article can be found online at https:// doi.org/10.1016/j.bmcl.2019.126909. References 1. (a) Galluzzi L, Kepp O, Vander Heiden MG, Kroemer G. Nat Rev Drug Discov.
5
(b) Xie H, Hanai J-I, Ren J-G, et al. Cell Metab. 2014;19:795. Cui W, Lv W, Qu Y, et al. Bioorg Med Chem Lett. 2016;26:3984. Boudreau A, Purkey HE, Hitz A, et al. Nat Chem Biol. 2016;12:779. Hou XM, Yuan SQ, Zhao D, Liu XJ, Wu XA. Biosci Rep. 2019;39 pii: BSR20181476. Di H, Zhang X, Guo Y, et al. Oncol Lett. 2018;15:5131. Rai G, Brimacombe KR, Mott BT, et al. J Med Chem. 2017;60:9184. Dragovich PS, Fauber BP, Corson LB, et al. Bioorg Med Chem Lett. 2013;23:3186. Fauber BP, Dragovich PS, Chen J, et al. Bioorg Med Chem Lett. 2013;23:5533. Ward RA, Brassington C, Breeze AL, et al. J Med Chem. 2012;55:3285. Lumeras W, Vidal L, Vidal B, et al. J Med Chem. 2011;54:7899.