2023, Vol. 34 
b Wuya College of Innovation, Key Laboratory of Structure-Based Drug Design & Discovery, Ministry of Education, Shenyang Pharmaceutical University, Shenyang, 110016, China;
c Institute of Structural Pharmacology & TCM Chemical Biology, College of Pharmacy, Fujian University of Traditional Chinese Medicine, Fuzhou 350122, China
Non-Hodgkin's lymphoma (NHL) is the most common hematological malignancy in adults. Approximately 4.3 million people worldwide suffer from this disease [1]. Diffuse large B-cell lymphoma (DLBCL) is the most common B-cell non-Hodgkin's lymphoma (B-NHL), accounting for roughly 30% [2,3]. Bruton's tyrosine kinase (BTK), as a member in BCR signaling pathway, plays a key role in the development, differentiation, survival, and signaling of B cells [4,5]. Therefore, signal transduction of BTK is crucial for the survival of B-cell leukemia and lymphoma [6,7].
Ibrutinib is the first oral BTK inhibitor approved by the FDA, which is used as a first-line treatment drug for various lymphomas, including CLL, MCL, FL, etc. [8]. Although ibrutinib has remarkable therapeutic effect, clinically drug-resistant cases still appeared with poor prognosis. The main mechanism of ibrutinib-resistance is BTKC481S mutation, which can also lead to the failure of other BTK covalent inhibitors, such as zanubrutinib and acalabrutinib [9,10]. Thus, there is an urgent need to develop new effective agents against BTK mutations.
Proteolysis-targeting chimeras (PROTAC) has recently become a research hot spot in academia and pharmaceutical industry [11,12]. However, PROTAC molecule has several intrinsic limitations, such as large molecular weight (roughly 900–1000 g/mol), lengthy linker [13]. These shortcomings may hinder further applications of PROTAC in clinical trials [14]. In 2018, we reported the first-generation of BTK degrader P13I [15,16]. However, P13I has a relatively large molecular weight with poor water solubility and moderate bioactivity. How to design the new structure of PROTACs while improving its bioactivity is a very challenging task.
In this study, we developed a new type of BTKC481S degrader L6 through computer-assisted design. Compared with P13I, molecular weight of L6 was reduced about 200, and the solubility and predicted permeability of L6 were improved. Moreover, the novel BTK degrader L6 has better bioactivity than P13I in ibrutinib-resistant BTKC481S HBL-1 cells (Fig. 1). In addition, we also conducted SAR study via molecular docking, providing a general procedure for further optimization of PROTACs.
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| Fig. 1. Development of novel PROTACs targeting ibrutinib-resistant BTKC481S. (A) Schematic representation and breakthrough of compound L6. (B) Design and evolutionary route of novel BTK degraders. | |
In order to optimize the structure and bioactivity of BTK degrader, we first designed derivatives of P13I with different E3 ligands and linkers. Then, ACD/Labs was employed for analysis. The results indicated that, BTK degraders with lenalidomide ligand could have better CaCo-2 cell membrane permeability than those with pomalidomide ligand (Table 1).
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Table 1 Chemical structures and parameters of novel BTK degraders and reference compound. |
Based on the analysis results above, we conducted the solubility test after synthesis of PROTACs (Scheme 1) [15]. Compound mL13I demonstrated a much better solubility than that of mP13I, indicated that lenalidomide ligand may also help to improve the solubility (Table 1). This phenomenon can also be found in compound L9I (Table 1). Therefore, lenalidomide was utilized as the E3 ligand moiety for BTK degrader development.
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| Scheme 1. Synthesis of compounds mL13I and L9I. | |
To further simplify the chemical structure of PROTACs, different BTK degraders with aliphatic linkers were prepared, and human Ramos cells and Mino cells were used to evaluate the degradation activity (Figs. S5 and S6 in Supporting information).
After the screening, we found that compound L6 with 6 carbon linker and lenalidomide ligand had the best BTK degradation activity at concentrations of 11, 33 and 100 nmol/L (Fig. 2 and Table S1 in Supporting information). Compared with P13I, L6 has a molecular weight reduction of about 200, and both hydrogen bond acceptors and rotatable bonds of L6 are reduced by 8. Additionally, the water solubility of L6 was about 3 times higher than that of P13I, and the predicted apparent permeability coefficient (Papp) of L6 in Caco-2 cells increased by nearly 10 times (Table 1).
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| Fig. 2. Structures and degradation efficiency of novel PROTACs with aliphatic linkers in B-cell malignancy Mino cells and Ramos cells. (A) Structures of the new generation of PROTAC molecules targeting BTK. (B) Immunoblotting analysis of BTK protein and β-actin protein from Ramos cells or Mino cells treated with compounds for 48 h. In 12-well plates, 2 × 105 cells were incubated in each well at 37 ℃. Grayscale analysis data was generated by ImageJ for the calculation of relative level of BTK protein. | |
For the synthesis of compounds mL13I and L9I (Scheme 1), the Sonogashira coupling reaction was conducted with compound 15 and 17 as substrate to prepare compounds 18 and 19. After reduction of alkynyl group, compound 21 and 22 were generated in which the hydroxy group was protected by p-toluenesulfonyl group. Compounds 23 and 24 were then utilized in substitution reaction to prepare compound 25 and 26. After the condensation reaction, ibrutinib derivatives were produced and then transformed to mL13I and L9I via click reaction in the last synthetic step. P13I and its derivatives were prepared as before [15]. For the synthesis of L6-L10 (Scheme 2), the intermediates B2 were synthesized via a Sonogashira coupling which was followed by the reduction of alkynyl group to generate alkyl acid ligand. Finally, compounds B4 were condensed with ibrutinib precursor to prepare the amide derivative as the target compounds L6-L10. Compared with P13I, mL13I, L9I and their derivatives, L6 has a pretty short synthetic route, which brings convenience for large-scale preparation in the future.
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| Scheme 2. Synthesis of compounds L6-L10. | |
In order to investigate the conformational differences between L6 and P13I in BTK-degrader-CRBN ternary complex, compounds were docked into BTK and CRBN structures. As showed in Fig. S10 (Supporting information), binding positions of L6 in two proteins were consistent with the crystal structures. Linker of P13I was curved, while the aliphatic linker of L6 was almost straight.
Polyethylene glycol linker may easily bind to H2O molecules. Therefore, the protein-protein interaction could be hindered. However, compound L6 is smaller and has fewer conformations; this may result in lower binding free energy and better stability of the ternary complex. Thus, efficiency of ubiquitination-degradation could be increased. To further confirm this phenomenon in experiments, we synthesized L7d with polyethylene glycol linker as derivative of L7. The results showed that bioactivity of L7d was reduced (Figs. S4 and S8 in Supporting information).
Next, to further evaluate the bioactivity of compound L6, different lymphoma cell lines were used. The data showed that L6 could efficiently degrade BTK protein in Ramos cell line (DC50 = 3.8 nmol/L, Fig. 3), which was nearly 5 times stronger than that of P13I (Figs. 4A and B). The inhibitory activity of L6 in ibrutinib-resistant BTKC481S HBL-1 cells was also better than that of P13I. Compared with ibrutinib, the EC50 of L6 was roughly 31 times better. Ibrutinib had nearly no efficacy to the mutant cell line (Fig. 4C and Table S2 in Supporting information) [10,17]. Therefore, L6 as a PROTAC molecule cannot only overcome the failure of ibrutinib caused by the BTKC481S mutation, but also improve the bioactivity of degraders.
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| Fig. 3. DC50 values of Western blotting analysis in B-cell malignancy Ramos cells and EC50 values of cell growth inhibition in ibrutinib-resistant HBL-1 (BTKC481S) cells. Immunoblotting analysis of BTK protein and β-actin protein from Ramos cells treated with compounds for 48 h. In 12-well plates, 2 × 105 cells were incubated in each well at 37 ℃. DC50 data was generated by GraphPad Prism 5. For cell viability assays, HBL-1 (BTKC481S) cells in 96-well plate were incubated for 72–96 h (3000 cells per well). The final EC50 was generated by MTT and GraphPad Prism 5. | |
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| Fig. 4. Bioactivities of novel BTKC481S degraders and reference compounds. (A) Relative protein level curves of BTK after treatment of Ramos cells with the indicated concentrations of compounds. (B) Immunoblotting analysis for BTK protein and β-actin protein from Ramos cells treated with the indicated concentrations of L6, L7, L8 and P13I for 48 h. (C) For cell viability assays, HBL-1 (BTKC481S) cells in 96-well plate were incubated for 72–96 h (3000 cells per well). The final EC50 was generated by MTT and GraphPad Prism 5. (D) For cell viability assays, DOHH2 cells in 96-well plate were incubated for 72–96 h (3000 cells per well). The final EC50 was generated by MTT and GraphPad Prism 5. | |
To further evaluate the general toxicity of compound L6, BTK-insensitive DOHH2 cell line was utilized. As showed in Fig. 4D, L6 had nearly no inhibitory activity even at 5000 nmol/L, indicated that L6 may serve as a safe and effective BTK degrader.
In summary, a novel ibrutinib-resistant BTKC481S degrader L6 with high efficiency and solubility was developed via computer-assisted optimization. Compound L6 has the lowest molecular weight among the current reported BTK degraders and better solubility than P13I. Compound L6 has fewer hydrogen bond donors/acceptors and rotatable bonds, and the predicted Papp in Caco-2 cells increased nearly 10 times compared with that of P13I. In terms of bioactivity, the newly developed L6 had a nearly 5-times increase in the degradation activity of BTK, and the inhibitory activity on ibrutinib-resistant BTKC481S HBL-1 cells was stronger than P13I. EC50 of L6 was about 31 times stronger than ibrutinib. Therefore, L6 not only represents an effective degrader with improved bioactivity to overcome the failure of ibrutinib, but also optimizes the structure and synthetic route of BTK PROTACs. Lastly, SAR study showed that the aliphatic linker in L6 had several advantages compared with polyethylene glycol linker of P13I, which provides a general and useful method for future development and optimization of PROTACs.
Declaration of competing interestThe 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.
AcknowledgmentsThis work was supported by the National Natural Science Foundation of China (Nos. 82125034, 81773567), National Major Scientific and Technological Project (Nos. 2020YFE0202200, 2021YFA1300200 and 2021YFA1302100), and Shuimu Tsinghua Scholar.
Supplementary materialsSupplementary material associated with this article can be found, in the online version, at doi:10.1016/j.cclet.2022.107924.
| [1] |
B.D. Cheson, J.P. Leonard, N. Engl. J. Med. 359 (2008) 613-626. DOI:10.1056/NEJMra0708875 |
| [2] |
L. Pasqualucci, D. Dominguez-Sola, A. Chiarenza, et al., Nature 471 (2011) 189-195. DOI:10.1038/nature09730 |
| [3] |
L.M. Morton, S.S. Wang, S.S. Devesa, et al., Blood 107 (2006) 265-276. DOI:10.1182/blood-2005-06-2508 |
| [4] |
J.C. Byrd, R.R. Furman, S.E. Coutre, et al., N. Engl. J. Med. 369 (2013) 32-42. DOI:10.1056/NEJMoa1215637 |
| [5] |
J.A. Burger, A. Wiestner, Nat. Rev. Cancer 18 (2018) 148-167. DOI:10.1038/nrc.2017.121 |
| [6] |
L.A. Honigberg, A.M. Smitha, M. Sirisawada, et al., Proc. Natl. Acad. Sci. U. S. A. 107 (2010) 13075-13080. DOI:10.1073/pnas.1004594107 |
| [7] |
W.H. Wilson, R.M. Young, R. Schmitz, et al., Nat. Med. 21 (2015) 922-926. DOI:10.1038/nm.3884 |
| [8] |
G. Li, X. Liu, X. Chen, Nat. Rev. Clin. Oncol. 17 (2020) 589-590. DOI:10.1038/s41571-020-0414-y |
| [9] |
J.A. Woyach, R.R. Furman, T. Liu, et al., N. Engl. J. Med. 370 (2014) 2286-2294. DOI:10.1056/NEJMoa1400029 |
| [10] |
R.R. Furman, S. Cheng, P. Lu, et al., N. Engl. J. Med. 370 (2014) 2352-2354. DOI:10.1056/NEJMc1402716 |
| [11] |
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 |
| [12] |
G.E. Winter, D.L. Buckley, J. Paulk, et al., Science 348 (2015) 1376-1381. DOI:10.1126/science.aab1433 |
| [13] |
A. Mullard, Nat. Rev. Drug Discov. 17 (2018) 777. |
| [14] |
X. Li, Y. Song, J. Hematol. Oncol. 13 (2020) 50. DOI:10.1002/jeq2.20016 |
| [15] |
Y. Sun, X. Zhao, N. Ding, et al., Cell Res. 28 (2018) 779-781. DOI:10.1038/s41422-018-0055-1 |
| [16] |
Y. Sun, N. Ding, Y. Song, et al., Leukemia 33 (2019) 2105-2110. DOI:10.1038/s41375-019-0440-x |
| [17] |
R.E. Davis, V.N. Ngo, G. Lenz, et al., Nature 463 (2010) 88-92. DOI:10.1038/nature08638 |