Hydrophobic tagging-induced degradation of NAMPT in leukemia cells (2024)

Nicotinamide phosphoribosyl transferase (NAMPT) is expressed in nearly all cells and involves in the biosynthesis nicotinamide adenine dinucleotide (NAD), a vital coenzyme for NAD-dependent enzyme (Fig. 1A) [1–5]. By regulating the intracellular NAD concentration, NAMPT orchestrates the critical biofunctions, such as adaptive responses to inflammatory, mitochondrial biogenesis and cellular metabolism [6–8]. However, NAMPT also implicates in the development of aging, obesity, nonalcoholic fatty liver disease (NAFLD) and type 2 diabetes mellitus (T2DM) [9–12]. Beyond these functions, NAMPT plays a pivotal role in the metabolism of many cancers including leukemia, colorectal, ovarian, breast, gastric, and so on [13]. In addition to its enzymatic function, extracellular NAMPT (eNAMPT) could also be secreted out of the cells and has cytokine-like activity [14,15]. Abnormal levels of eNAMPT engaged in tumor progression by modulating angiogenesis, remodeling tumor immune microenvironment, and promoting the proliferation, migration, and epithelial–mesenchymal transition (EMT) of tumor cells [3,16,17].

Figure 1

It has been demonstrated that the enzyme activity of NAMPT can be inhibited by small-molecule inhibitors. A few NAMPT specific inhibitors, such as OT-82, CHS828 and FK866, have entered clinical trials for cancer therapy [18,19]. Despite encouraging preclinical evidence of the potential utility of these inhibitors in cancer models, none of them is able to progress to the later stages of clinical trials owing to the limited anti-tumor efficacy [13,20]. One possible reason is that the inhibition of intracellular NAMPT enzymatic activity may not be sufficient to fully impair the oncogenic function of NAMPT because the roles of eNAMPT are enzymatically-independent [14]. Therefore, the adaptation of additional tactics to boost the efficacy of NAMPT blockage is highly desirable.

Targeted protein degradation (TPD), represented by proteolysis-targeting chimera (PROTAC), is an emerging therapeutic strategy in drug development [21–24]. Although PROTACs have numerous advantages such as catalytic mechanism, avoiding drug resistance and tackling undruggable targets, clinical development of PROTACs is still limited by high molecular weight, poor cellular permeability, unfavorable pharmaco*kinetics, and low bioavailability [25]. Hydrophobic tagging (HyT) technology presents an alternative strategy for TPD, which relatively has lower molecular weight and reduced the elimination of potential teratogenic side effects associated with E3 ligase ligands (e.g. thalidomide-derived cereblon ligands) [26,27].

A typical HyT molecule consists of an identifying peptide or small-molecule ligand that specifically binds to the protein of interest (POI), and a hydrophobic tag that mimics a misfolded protein state, thus leading to the degradation of protein by recruiting chaperones or proteasomes [28,29]. A small number of hydrophobic tags (e.g. adamantane and fluorene) have been discovered and successfully employed in the design of HyT degraders [27]. However, the target scope, pharmacological activity and mechanism of action remain largely unexplored. Previously, we confirmed that targeted protein degradation was an effective strategy to interfere with the function of NAMPT and designed the effective NAMPT degraders using the PROTAC approach [30,31]. Nevertheless, it is still unknown whether NAMPT could be effectively degraded by the HyT strategy. Herein, we designed the first HyT-mediated NAMPT degraders, which showed potent NAMPT degrading potency and selective anti-leukemia ability.

Previously, we identified potent NAMPT inhibitors (MS7 and MS0) through high-throughput screening and structure-based drug design [32]. These compounds shown excellent in vitro inhibitory activity against NAMPT, offering favorable ligands for the design of NAMPT degraders. Binding mode analysis of MS7 with NAMPT revealed that the benzyl piperazine was exposed to the solvent (Fig. 1B). Therefore, various linkers and HyT tags were introduced on the benzyl or piperazine group, affording compounds NH-1–NH-13 (Fig. 1C). To further explore the effects of NAMPT ligands on the degrading activity, our previously identified inhibitor MS35 [33] was also used to design HyT-based degraders NH-16–NH-24 (Fig. 1C).

Chemical synthesis of the target compounds NH-1–NH-13 was depicted in Schemes S1 and S2 (Supporting information). 4-Nitrobenzenesulfonyl chloride underwent substitution reaction with tert-butyl piperazine-1-carboxylate under basic conditions to obtain compound 3. The nitro group of compound 3 was then reduced to the amino group in the presence of Pd/C and H₂, and the resulting product (compound 4) was further reacted with sodium azide and 3-pyridylamine separately to obtain urea compound 6. Compound 6 was deprotected by trifluoroacetic acid (TFA) to remove the Boc protecting group, resulting in key intermediate 7 (MS0). Compound 7 underwent substitution with methyl-4-(bromomethyl)benzoate, followed by the removal of the ester protecting group, to afford key intermediate 9 (MS7). Subsequently, different lengths of fatty chains were connected to adamantane to obtain linker containing hydrophobic tags 12a-b, 15a-d, 17, and 19a-e. These products were then coupled with key intermediate 7 or 9 to form target compounds NH-1–NH-12. Key intermediate 7 was also connected to the linker tags and then coupled with norbornene to obtain the target compound NH-13. The synthetic routes for target compounds NH-14–NH-24 were shown in Schemes S3 and S4 (Supporting information). 4-Ethynylbenzoic acid and 3-(aminomethyl)pyridine were coupled under the N, N, N′, N′-tetramethyl-O-(1H-benzotriazol-1-yl)uronium hexafluorophosphate (HBTU) and Et₃N conditions to obtain intermediate 24. Intermediate 24 underwent click reaction with intermediate 25 to obtain key intermediate 26. After hydrolysis, key intermediate 26 was connected to different linker tags, and then coupled with norbornene or adamantane group to obtain target compounds NH-14–NH-24.

Initially, the NAMPT inhibition activity of the designed HyT degraders were assayed to confirm whether they could bind with NAMPT binding. The results indicated that most compounds effectively inhibited NAMPT with half-maximal inhibitory concentration (IC₅₀) values in the nanomolar range except for compounds NH-6, NH-20 and NH-21 (Fig. 1D). Then, the NAMPT degradation ability of compounds NH-1–NH-12 was detected by the Western blot analysis in A2780 cells, an ovarian cancer cell line that was widely used in the NAMPT degradation assay [34]. However, there was no obvious degradation effect on this cell line after exposure with 1 or 10 µmol/L compounds for 24 h (Fig. 2A). Thus, we further investigated the NAMPT degradation activity on various cancer cell lines including colon cancer (HCT-116), cervical cancer (HeLa), and leukemia cells (K562, Jurkat, HL60). Interestingly, significant degradation was only observed in leukemia cells, particularly for K562 and HL60 cells (Figs. 2B and C). Generally, MS0-based HyT degraders (NH-7–NH-12) were more potent than MS7-based HyT degraders (NH-1–NH-6), which also have relatively lower molecular weight. Among these compounds, NH-11 showed the best NAMPT inhibitory and degrading activity. When the NAMPT ligand was replaced by MS35, the resulting compound (NH-22) had less activity to induce NAMPT degradation than compound NH-11. Recently, norbornene was discovered to be an effective HyT tag and was successfully employed in the design of HyT degraders targeting anaplastic lymphoma kinase and intractable enhancer of zeste hom*olog 2 [27]. Inspired by that, we replaced the HyT part of NH-11 with norbornene and obtained NH-13. However, the NAMPT degrading activity was significantly decreased. We further synthesized norbornene tagged degraders NH-16–NH-22 and NH-23–NH-24 on the basis of NAMPT inhibitor MS35 (Fig. 2D).

Figure 2

NH-11 dose-dependently induced NAMPT degradation with DC₅₀ values of 3.18 µmol/L (K562), 6.05 µmol/L (HL60) and 7.32 µmol/L (Jurkat), respectively (Figs. 3A and B). The DC₅₀ values of NH-13 on K562, Jurkat and HL60 cells were 10.34, 10.47 and 10.80 µmol/L, respectively (Fig. 3A and Fig. S1A). Compound NH-16 had DC₅₀ values of 10.21, 11.71 and 12.11 µmol/L against K562, Jurkat and HL60 cells, respectively. NH-22 had DC₅₀ values of 9.75 µmol/L on K562, 9.10 µmol/L on Jurkat, and 11.21 µmol/L on HL60, respectively (Fig. 3C, Figs. S1B and C). Additionally, the NAMPT degradation induced by compound NH-11 was also dependent on the duration of exposure. Following incubation with 8 µmol/L NH-11 for 48 h, a maximum degradation of approximately 95% of NAMPT protein was observed (Fig. 3D). These results indicated that NH-11 might be the lead compound for NAMPT degradation in leukemia cells.

Figure 3

Compound NH-11 possessed the highest capacity for NAMPT degradation and was selected for further mechanism and antitumor activity evaluations. In eukaryotic cells, damaged proteins can be degraded by proteasomes or lysosomes [35,36]. To investigate the potential degradation mechanism of NH-11, we performed rescue assays. The K562 cells was co-treated with NH-11 and lysosomal inhibitor bafilomycin A1 (Baf A1) [37], or ubiquitin (Ub)-proteasome inhibitors MG132 [38], carfilzomib [39]. It was found that the MG132 and carfilzomib largely abolished NH-11-induced NAMPT degradation (Fig. 3E), indicating that NH-11 mediated the degradation of NAMPT via the Ub-proteasome system (UPS). Given the potent NAMPT inhibition activity and degradation efficiency of NH-11, its antiproliferative activity against leukemia cell lines were also investigated by using a cell counting kit-8 (CCK8) assay. The results revealed that NH-11 showed potent antiproliferative activity against K562, Jurkat and HL60 cells with IC₅₀ value of 0.73 ± 0.31, 0.03 ± 0.01, and 0.95 ± 0.38 µmol/L, respectively (Fig. S2 and Table S2 in Supporting information).

We next investigated the mechanism of NH-11 in inhibiting proliferation of leukemia cells. The Annexin V-FITC and propidium iodide (PI) staining assay indicated that NH-11 significantly induced apoptosis of K562 cells after 48 h of treatment and the apoptosis rate was enhanced in a dose-dependent manner (Figs. 4A and C). In addition, flow cytometric analysis was conducted to investigate the effects of compound NH-11 on the cell cycle of K562 cells. After treatment with 1 or 5 µmol/L of degrader NH-11 for 48 h, 37.42% and 26.71% of K562 cell remained in the G1 stage, which was significantly lower than that in the DMSO treatment group. Notably, after treatment with 5 µmol/L of degrader NH-11 for 48 h, 52.05% of K562 cells showed a prominent cell cycle arrest in the S phase (Figs. 4B and D). Moreover, consistent with the flow cytometric assay, NH-11 could sustain the downregulation of Bcl-2, a key anti-apoptotic protein, whereas it could increase the levels of Bax, a protein promotes cell death (Figs. 4E and F). Finally, NAD⁺/NAMPT activity was also detected. Compared with inhibitor MS7, degrader NH-11 could more effectively inhibit the synthesis of NAD by NAMPT (IC₅₀ = 35.71 ± 2.09 nmol/L, Fig. 4G). In addition, the cytotoxicity of NH-11 on normal cell lines L02 and MCF-10A was also evaluated. The results indicated that there was no obvious cytotoxic effect on normal cells (Fig. 4H). Taken together, these results suggested that NH-11 effectively and selectively degraded NAMPT in K562 cells and then induced the cell apoptosis.

Figure 4

In summary, a series of novel HyT NAMPT degraders were designed and synthesized by connecting hydrophobic tags adamantane and norbornene to several NAMPT inhibitors. HyT degrader NH-11 selectively degraded NAMPT in leukemia cells, blocking the secretion of intracellular NAMPT. Thus, we hypothesized that the level of eNAMPT could be reduced. Compound NH-11 showed potent antiproliferative activities and effectively induced cell apoptosis and cell cycle arrest in leukemia cells. Comparing with reported NAMPT PROTAC degraders, NH-11 possesses relatively lower molecular weight and good selectivity for leukemia cells. Thus, the hydrophobic tag may be an alternative NAMPT degradation strategy and further optimizations are currently in progress.

Declaration of competing interest

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.

Acknowledgments

This work was supported by the National Key Research and Development Program of China (No. 2022YFC3401500 to C. S.), the National Natural Science Foundation of China (No. 82030105 to C. S.), and Shanghai Rising-Star Program (No. 20QA1411700 to G. D.). We thank the staff members of the Large-scale Protein Preparation System at the National Facility for Protein Science in Shanghai (NFPS), Shanghai Advanced Research Institute, Chinese Academy of Science, China for providing technical support and assistance in data collection and analysis.

Supplementary materials

Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.cclet.2023.109392.

1. [1]

   B. Samal, Y. Sun, G. Stearns, et al., Mol. Cell. Biol. 14 (1994) 1431–1437.

2. [2]

   D. Friebe, M. Neef, J. Kratzsch, et al., Diabetologia 54 (2011) 1200–1211. doi: 10.1007/s00125-010-2042-z

3. [3]

   A. Rongvaux, R.J. Shea, M.H. Mulks, et al., Eur. J. Immunol. 32 (2002) 3225–3234. doi: 10.1002/1521-4141(200211)32:11<3225::AID-IMMU3225>3.0.CO;2-L

4. [4]

   J.R. Revollo, A.A. Grimm, S. Imai, J. Biol. Chem. 279 (2004) 50754–50763. doi: 10.1074/jbc.M408388200

5. [5]

   A. Garten, S. Schuster, M. Penke, et al., Nat. Rev. Endocrinol. 11 (2015) 535–546. doi: 10.1038/nrendo.2015.117

6. [6]

   V. Audrito, V.G. Messana, S. Deaglio, Front. Oncol. 10 (2020) 358. doi: 10.3389/fonc.2020.00358

7. [7]

   J.S. Kim, C.S. Yoon, D.R. Park, J. Exerc. Nutrit. Biochem. 18 (2014) 259–266. doi: 10.5717/jenb.2014.18.3.259

8. [8]

   R. Manickam, J. Tur, S.L. Badole, et al., J. Cach. Sarcop. Muscle 13 (2022) 1177–1196. doi: 10.1002/jcsm.12887

9. [9]

   S. Imai, L. Guarente, Trends Cell Biol. 24 (2014) 464–471. doi: 10.1016/j.tcb.2014.04.002

10. [10]

    S. Aggarwal, N. Trehanpati, P. Nagarajan, G. Ramakrishna, J. Cell. Physiol. 237 (2022) 3164–3180. doi: 10.1002/jcp.30772

11. [11]

    E. van der Veer, C. Ho, C. O'Neil, et al., J. Biol. Chem. 282 (2007) 10841–10845. doi: 10.1074/jbc.C700018200

12. [12]

    F.F. Sheng, X.P. Dai, J. Qu, et al., Clin. Exp. Pharmacol. Physiol. 38 (2011) 550–554. doi: 10.1111/j.1440-1681.2011.05548.x

13. [13]

    U. Galli, C. Travelli, A. Massarotti, et al., J. Med. Chem. 56 (2013) 6279–6296. doi: 10.1021/jm4001049

14. [14]

    A.A. Grolla, C. Travelli, A.A. Genazzani, J.K. Sethi, Br. J. Pharmacol. 173 (2016) 2182–2194. doi: 10.1111/bph.13505

15. [15]

    S. Torretta, G. Colombo, C. Travelli, et al., Cells 9 (2020) 496. doi: 10.3390/cells9020496

16. [16]

    R. Adya, B.K. Tan, A. Punn, J. Chen, H.S. Randeva, Cardiovasc. Res. 78 (2008) 356–365. doi: 10.1093/cvr/cvm111

17. [17]

    F. Carbone, L. Liberale, A. Bonaventura, et al., Compr. Physiol. 7 (2017) 603–621.

18. [18]

    Y. Wei, H. Xiang, W. Zhang, Front. Pharmacol. 13 (2022) 970553. doi: 10.3389/fphar.2022.970553

19. [19]

    U. Galli, G. Colombo, C. Travelli, et al., Front. Pharmacol. 11 (2020) 656. doi: 10.3389/fphar.2020.00656

20. [20]

    L. Korotchkina, D. Kazyulkin, P.G. Komarov, et al., Leukemia 34 (2020) 1828–1839. doi: 10.1038/s41375-019-0692-5

21. [21]

    K.M. Sakamoto, K.B. Kim, A. Kumagai, et al., Proc. Natl. Acad. Sci. U. S. A. 98 (2001) 8554–8559. doi: 10.1073/pnas.141230798

22. [22]

    Y. Sun, X. Luo, Z. Yang, et al., Chin. Chem. Lett. 34 (2023) 107924. doi: 10.1016/j.cclet.2022.107924

23. [23]

    A. Chen, Y. Zhong, Y. Liu, et al., Chin. Chem. Lett. 34 (2023) 107923. doi: 10.1016/j.cclet.2022.107923

24. [24]

    M. Ao, J. Wu, Y. Cao, et al., Chin. Chem. Lett. 34 (2023) 107741. doi: 10.1016/j.cclet.2022.107741

25. [25]

    T.K. Neklesa, J.D. Winkler, C.M. Crews, Pharmacol. Ther. 174 (2017) 138–144. doi: 10.1016/j.pharmthera.2017.02.027

26. [26]

    G.E. Winter, D.L. Buckley, J. Paulk, et al., Science 348 (2015) 1376–1381. doi: 10.1126/science.aab1433

27. [27]

    S. Xie, F. Zhan, J. Zhu, et al., Angew. Chem. Int. Ed. 62 (2023) e202217246. doi: 10.1002/anie.202217246

28. [28]

    Y. Wang, X. Jiang, F. Feng, W. Liu, H. Sun, Acta Pharm. Sin. B 10 (2020) 207–238. doi: 10.1016/j.apsb.2019.08.001

29. [29]

    L.M. Luh, U. Scheib, K. Juenemann, et al., Angew. Chem. Int. Ed. 59 (2020) 15448–15466. doi: 10.1002/anie.202004310

30. [30]

    Y. Wu, C. Pu, Y. Fu, et al., Acta Pharm. Sin. B 12 (2022) 2859–2868. doi: 10.1016/j.apsb.2021.12.017

31. [31]

    J. Cheng, S. He, J. Xu, et al., J. Med. Chem. 65 (2022) 15725–15737. doi: 10.1021/acs.jmedchem.2c01243

32. [32]

    T.Y. Xu, S.L. Zhang, G.Q. Dong, et al., Sci. Rep. 5 (2015) 10043. doi: 10.1038/srep10043

33. [33]

    G. Dong, W. Chen, X. Wang, et al., J. Med. Chem. 60 (2017) 7965–7983. doi: 10.1021/acs.jmedchem.7b00467

34. [34]

    V. Tolstikov, A. Nikolayev, S. Dong, G. Zhao, M.S. Kuo, PLoS One 9 (2014) e114019. doi: 10.1371/journal.pone.0114019

35. [35]

    C. de Duve, Nat. Cell Biol. 7 (2005) 847–849. doi: 10.1038/ncb0905-847

36. [36]

    I. Dikic, Annu. Rev. Biochem. 86 (2017) 193–224. doi: 10.1146/annurev-biochem-061516-044908

37. [37]

    H.W.S. Tan, B. Anjum, H.M. Shen, et al., Autophagy 15 (2019) 1455–1459. doi: 10.1080/15548627.2019.1609847

38. [38]

    Y. Sun, X. Zhao, N. Ding, et al., Cell Res. 28 (2018) 779–781. doi: 10.1038/s41422-018-0055-1

39. [39]

    K. Raina, J. Lu, Y. Qian, et al., Proc. Natl. Acad. Sci. U. S. A. 113 (2016) 7124–7129. doi: 10.1073/pnas.1521738113